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EP1155321A1 - Compositions and methods for monitoring the modification of engineered binding partners - Google Patents

Compositions and methods for monitoring the modification of engineered binding partners

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Publication number
EP1155321A1
EP1155321A1 EP00906478A EP00906478A EP1155321A1 EP 1155321 A1 EP1155321 A1 EP 1155321A1 EP 00906478 A EP00906478 A EP 00906478A EP 00906478 A EP00906478 A EP 00906478A EP 1155321 A1 EP1155321 A1 EP 1155321A1
Authority
EP
European Patent Office
Prior art keywords
binding
binding domain
enzyme
binding partner
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00906478A
Other languages
German (de)
French (fr)
Inventor
John Colyer
Derek Woolfson
Roger Kingdon Craig
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cyclacel Ltd
Original Assignee
Cyclacel Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cyclacel Ltd filed Critical Cyclacel Ltd
Publication of EP1155321A1 publication Critical patent/EP1155321A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the invention relates to monitoring of the post-translational modification of a protein.
  • the post-translational modification of proteins have been known for over 40 years and since then has become a ubiquitous feature of protein structure.
  • the addition of biochemical groups to translated polypeptides has wide-ranging effects on protein stability, protein secondary/tertiary structure, enzyme activity and in more general terms on the regulated homeostasis of cells.
  • Such modifications include, but are not limited to, the addition of a phosphate (phosphorylation), carbohydrate (glycosylation), ADP-ribosyl (ADP ribosylation), fatty acid (prenylation, which includes but is not limited to: myristoylation and palmitylation), ubiquitin (ubiquitination) and sentrin (sentrinization; a ubiquitination-like protein modification).
  • Additional examples of post-translational modification include methylation, actylation, hydroxylation, iodination and flavin linkage. Many of the identified modifications have important consequences for the activity of those polypeptides so modified.
  • Phosphorylation is a well-studied example of a post-translational modification of protein.
  • polypeptides form higher order tertiary structures with like polypeptides (homo-oligomers) or with unlike polypeptides (hetero-oligomers).
  • homo-oligomers like polypeptides
  • hetero-oligomers unlike polypeptides
  • two identical polypeptides associate to form an active homodimer.
  • An example of this type of association is the natural association of myosin II molecules in the assembly of myosin into filaments.
  • the dimerization of myosin II monomers is the initial step in seeding myosin filaments.
  • the initial dimerization is regulated by phosphorylation the effect of which is to induce a conformational change in myosin II secondary structure resulting in the folded 10S monomer subunit extending to a 6S molecule.
  • This active molecule is able to dimerize and subsequently to form filaments.
  • the involvement of phosphorylation of myosin II in this priming event is somewhat controversial. Although in higher eukaryotes the conformational change is dependent on phosphorylation, in Ancanthoamoeba, a lower eukaryote, the post- translational addition of phosphate is not required to effect the initial dimerization step.
  • the dimerization domains in myosin II of higher eukaryotes contain the sites for phosphorylation and it is probable that phosphorylation in this region is responsible for enabling myosin II to dimerize and subsequently form filaments.
  • Dictyostelium this situation is reversed in that the phosphorylation sites are outside the dimerization domain and phosphorylation at these sites is required to effect the disassembly of myosin filaments.
  • Acanthoamoeba myosin II is phosphorylated in the dimerization domain but this modification is not necessary to enable myosin II monomers to dimerize in this species.
  • post-translational modification is the addition of phosphate to polypeptides by specific enzymes known as protein kinases.
  • protein kinases enzymes that have been identified as important regulators of the state of phosphorylation of target proteins and have been implicated as major players in regulating cellular physiology.
  • the cell-division-cycle of the eukaryotic cell is primarily regulated by the state of phosphorylation of specific proteins the functional state of which is determined by whether or not the protein is phosphorylated. This is determined by the relative activity of protein kinases which add phosphate and protein phosphatases which remove the phosphate moiety from these proteins.
  • dysfunction of either the kinases or phosphatases may lead to a diseased state.
  • the regulatory pathway is composed of a large number of genes that interact in vivo to regulate the phosphorylation cascade that ultimately determines if a cell is to divide or arrest cell division.
  • labeled for example, radiolabeled
  • labeled moieties e.g., phosphate, fatty acyl (including, but not limited to, myristoyl, palmityl, sentrin, methyl, actyl, hydroxyl, iodine, flavin, ubiquitin or ADP-ribosyls
  • Analysis of modified proteins is typically performed by electrophoresis and autoradiography, with specificity enhanced by immunoprecipitation of proteins of interest prior to electrophoresis.
  • Back-labeling The enzymatic incorporation of a labeled (including, but not limited to, with a radioactive and fluorescent label) moiety into a protein in vitro to estimate the state of modification in vivo.
  • cell-membrane-permeable protein-modifying enzyme inhibitors e.g., Wortmannin, staurosporine
  • Antibody recognition of the modified form of the protien e.g., using an antibody directed at ubiquitin or carbohydrate epitopes, e.g., by Western blotting, of either 1- or 2- dimensional gels bearing test protein samples.
  • kinase inhibitors have adequate specificity to allow for the unequivocal correlation of a given kinase with a specific kinase reaction. Indeed, many inhibitors have a broad inhibitory range.
  • staurosporine is a potent inhibitor of phospholipid/Ca +2 dependant kinases.
  • Wortmannin is some what more specific, being limited to the phosphatidylinositol-3 kinase family. This is clearly unsatisfactory because more than one biochemical pathway may be affected during treatment making the assignment of the effects almost impossible.
  • yeast Sacharomyces cervisiae and Schizosaccharomyces pombe
  • yeast has been exploited as a model organism for the identification of gene function using recessive mutations. It is through research on the effects of these mutations that the functional specificities of many protein-modifying enzymes have been elucidated.
  • these molecular genetic techniques are not easily transferable to higher eukaryotes, which are diploid and therefore not as genetically tractable as these lower eukaryotes.
  • Ras proteins are a conserved group of polypeptides located at the plasma membrane which exist in either a GTP-bound active state or in a GDP-bound inactive state. This family of proteins operates in signal transduction pathways that regulate cell growth and differentiation. In higher eukaryotes, Ras is a key regulator that mediates signal transduction from cell surface tyrosine kinase receptors to the nucleus via activation of the MAP kinase cascade.
  • Ras directly binds a serine/threonine kinase, Raf-1, a product of the c-raf-1 proto-oncogene, and that this association leads to stimulation of the activity of Raf-1 to phosphorylate MAP kinase kinase (MEK).
  • MEK MAP kinase kinase
  • Another post-translational modification is the addition of ubiquitin to selected polypeptides. This provides a key mechanism by which to control the abundance of important regulatory proteins, for example, Gl and mitotic cyclins and the p53 tumor suppressor protein.
  • Ubiquitin is a highly conserved 76-amino-acid cellular polypeptide.
  • ubiquitin The role of ubiquitin in targeting proteins for degradation involves the specific ligation of ubiquitin to the ⁇ group of lysine residues in proteins that are to be degraded or internalized from the plasma membrane.
  • the ubiquitin tag determines the fate of the protein and results in its selective proteolysis. Recently a number of factors have been isolated and shown to be involved in the ubiquitination process.
  • the initial step in the addition of ubiquitin to a protein is the activation of ubiquitin by the ubiquitin activating enzyme, El .
  • This is an ATP-dependent step resulting in the formation of a thioester bond between the carboxyl terminal glycine of ubiquitin and the active site cysteine residue of El .
  • Activated ubiquitin then interacts with a second factor, the E2 protein.
  • a thioester bond forms between the activated glycine residue of ubiquitin and a cysteine residue in a specific E2 protein.
  • the E2 proteins represent a family of closely- related proteins encoded by different genes that confer specificity in the proteolytic process.
  • E3 completes the final step of ubiquitination by attaching ubiquitin via the ⁇ amino group on lysine residues in proteins to be targeted for degradation. Moreover, E3 is able to add ubiquitin to ubiquitin molecules already attached to target proteins, thereby resulting in polyubiquitinated proteins that are ultimately degraded by the multi-subunit proteasome.
  • heterodimer association is described in patent application number WO92/00388. It describes an adenosine 3: 5 cyclic monophosphate (cAMP) dependent protein kinase which is a four-subunit enzyme being composed of two catalytic polypeptides (C) and two regulatory polypeptides (R). In nature the polypeptides associate in a stoichiometry of R 2 C 2 . In the absence of cAMP the R and C subunits associate and the enzyme complex is inactive. In the presence of cAMP the R subunit functions as a ligand for cAMP resulting in dissociation of the complex and the release of active protein kinase. The invention described in WO92/00388 exploits this association by adding fluorochromes to the R and C subunits.
  • cAMP cyclic monophosphate
  • the polypeptides are labeled (or 'tagged') with fluorophores having different excitation/emission wavelengths.
  • the emission from one such fluorophore following excitation effects a second excitation/emission event in the second fluorophore.
  • concentration of cAMP By monitoring the fluorescence emission or absorption of each fluorophore, which reflects the presence or absence of fluorescence energy transfer between the two, it is possible to derive concentration of cAMP as a function of the association between the R and C subunits. Therefore, the natural affinity of the C subunit for the R subunit has been exploited to monitor the concentration of a specific metabolite, namely cAMP.
  • Tsien et al. (WO97/28261) teach that fluorescent proteins having the proper emission and excitation spectra that are brought into physically close proximity with one another can exhibit fluorescence resonance energy transfer ("FRET").
  • FRET fluorescence resonance energy transfer
  • the invention of WO97/28261 takes advantage of that discovery to provide tandem fluorescent protein constructs in which two fluorescent protein labels capable of exhibiting FRET are coupled through a linker to form a tandem construct.
  • protease activity is monitored using FRET to determine the distance between fluorophores controlled by a peptide linker and subsequent hydrolysis thereof.
  • Other applications rely on a change in the intrinsic fluorescence of the protein as in the kinase assays of WO98/06737.
  • the present invention instead encompasses the use of FRET or other detection procedures to monitor the association of polypeptides, as described herein, which are labeled with fluorescent labels (protein and chemical); in the invention, FRET, fluorescence correlation spectroscopy, fluorescence anisotropy, monome ⁇ excimer fluorescence or other techniques indicate the proximity of two labeled polypeptide binding partners, which labeled partners associate either in the presence or absence of a given post-translational modification to an engineered site which has been introduced into at least one of the partners, but not into the fluorophore, reflecting the modification state of one or both of the binding partners and, consequently, the level of activity of a protein-modifying enzyme.
  • the invention further provides methods which employ non-fluorescent labels including, but not limited to, radioactive labels.
  • the invention encompasses methods which do not employ detectable labels; such methods include, but are not limited to, the detection of the inhibition or reconstitution of enzymatic activity, which inhibition or reconstitution results from modification-dependent binding or dissociation between an engineered binding domain and a binding partner therefor.
  • the invention provides engineered binding domains, sequences and polypeptides, all as defined below, as well as kits comprising these molecules and assays of enzymatic function in which they are employed as reporter molecules.
  • One aspect of the invention is an isolated engineered binding domain and a binding partner therefor, wherein the isolated engineered binding domain includes a site for post- translational modification and binds the binding partner therefor in a manner dependent upon modification of the site.
  • the invention additionally encompasses a method for monitoring activity of an enzyme comprising performing a detection step to detect binding of an isolated engineered binding domain and a binding partner therefor as a result of contacting one or both of the isolated engineered binding domain and the binding partner with the enzyme, wherein the isolated engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site and wherein detection of binding of the isolated engineered binding domain and the binding partner as a result of the contacting is indicative of enzyme activity.
  • Another aspect of the invention is a method for monitoring activity of an enzyme comprising performing a detection step to detect dissociation of an isolated engineered binding domain from a binding partner therefor as a result of contacting one or both of the isolated engineered binding domain and the binding partner with the enzyme, wherein the isolated engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site and wherein detection of dissociation of the isolated engineered binding domain from the binding partner as a result of the contacting is indicative of enzyme activity.
  • binding domain refers in a three-dimensional sense to the amino acid residues of a first polypeptide required for modification-dependent binding between the first polypeptide and its binding partner.
  • the amino acids of a "binding domain” may be either contiguous or non-contiguous.
  • a binding domain must include at least 1 amino acid, and may include 2 or more, preferably 4 or more, amino acids which are contiguous or non-contiguous, but are necessary for modification-dependent binding to the binding partner, and may include a full-length protein.
  • a binding domain of use in the invention may be present on a polypeptide chain that consists solely of the binding domain amino acid sequence or may be present in the context of a larger polypeptide molecule (i.e., one which comprises amino acids other than those of the binding domain), which molecule may be either naturally-occurring or recombinant and, in the case of the latter, may comprise either natural or non-natural amino acid sequences.
  • engineered binding domain refers to a binding domain, as defined above, which is an amino acid sequence that is altered (i.e., by insertion, deletion or substitution of at least one amino acid) such that the domain amino acid sequence is no longer as found in nature. The position of the altered amino acid is within the residues which form the domain.
  • An engineered binding domain of use in the invention may be present on a polypeptide chain that consists solely of the engineered binding domain amino acid sequence or may be present in the context of a larger polypeptide molecule (i.e., one which comprises amino acids other than those of the engineered binding domain), which molecule may be either naturally-occurring or recombinant and, in the case of the latter, may comprise either natural or non-natural amino acid sequences.
  • sites for post-translational modification may be present in either or both of the engineered domain and its binding partner, as defined above. If such sites are present on both the engineered domain and its binding partner, binding between the domain and its partner may be dependent upon the modification state of either one or both sites. If a single polypeptide chain comprises the engineered domain and its binding partner (or two engineered binding domains), the state of modification of one or both sites will determine whether binding between the two domains occurs.
  • site and “site sufficient for the addition of refer to an amino acid sequence which is recognized by (i.e., a signal for) a modifying enzyme for the purpose of post-translational modification (i.e., addition or removal of a "moiety” as defined below) of the polypeptide or a portion thereof.
  • a “site” additionally refers to the single amino acid which is modified. It is contemplated that a site comprises a small number of amino acids, as few as one but typically from 2 to 10, less often up to 30 amino acids, and further that a site comprises fewer than the total number of amino acids present in the polypeptide.
  • a "site”, for post-translational modification is present on an engineered binding domain and, optionally, its ' binding partner, as defined below. If such sites are present on both engineered binding domain and the binding partner, binding between them may be dependent upon the modification state of either one or both sites. If a single polypeptide chain comprises the engineered binding domain and its binding partner (or two engineered binding domains), the state of post-translational modification of one or both sites will determine whether binding between the engineered binding domain and the binding partner occurs.
  • modification refers to the addition or removal of a chemical "moiety", as described herein, to/from a site on a polypeptide chain and does not refer to other post-translational events which do not involve addition or removal of such a moiety as described herein, and thus does not include simple cleavage of the reporter molecule polypeptide backbone by hydrolysis of a peptide bond, but does include hydrolysis of an isopeptide bond (e.g., in the removal of ubiquitin).
  • moiety and “group” refer to one of the post-translationally added or removed groups referred to herein: i.e., one of a phosphate, ubiquitin, glycosyl, fatty acyl, sentrin or ADP-ribosyl moiety.
  • binding partner refers to a polypeptide or fragment thereof (a peptide) that binds to a binding domain, as defined herein, in a manner which is dependent upon the state of modification of a site for post-translational modification which is, at a minimum, present upon the binding domain; the binding partner itself may, optionally, comprise such a site and binding between the binding domain and its corresponding binding partner may, optionally, depend upon modification of that site.
  • a binding partner does not necessarily have to contain a site for post-translational modification if such a site is not required to be present on it for modification-dependent association between it and a binding domain.
  • the term “isolated” refers to a molecule or population of molecules that is substantially pure (i.e., free of contaminating molecules of unlike amino acid sequence).
  • polypeptide and peptide refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.
  • Polypeptide D refers to either a full-length naturally-occurring amino acid chain or a "fragment thereof or "peptide", such as a selected region of the polypeptide that is of interest in a binding assay and for which a binding partner is known or determinable, or to an amino acid polymer, or a fragment or peptide thereof, which is partially or wholly non-natural.
  • ""Fragment thereof thus refers to an amino acid sequence that is a portion of a full-length polypeptide. between about 8 and about 500 amino acids in length, preferably about 8 to about 300, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids in length.
  • Protein refers to a short amino acid sequence that is 10-40 amino acids long, preferably 10-35 amino acids. Additionally, unnatural amino acids, for example, ⁇ -alanine, phenyl glycine and homoarginine may be included. Commonly- encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L- optical isomer. The L-isomers are preferred. In addition, other peptidomimetics are also useful, e.g. in linker sequences of polypeptides of the present invention (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267).
  • protein refers to a linear sequence of amino acids which exhibits biological function.
  • This linear sequence includes full-length amino acid sequences (e.g. those encoded by a full-length gene or polynucleotide), or a portion or fragment thereof, provided the biological function, as well as the post-translational- modification-dependent binding function, is maintained by that portion or fragment.
  • subunit and domain also may refer to polypeptides and peptides having biological function.
  • a peptide useful in the invention will at least have a binding capability, i.e, with respect to binding as or to a binding partner, and also may have another biological function that is a biological function of a protein or domain from which the peptide sequence is derived.
  • Polynucleotide refers to a polymeric form of nucleotides of at least 10 bases in length and up to 1 ,000 bases or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • substantially refers to that which is at least 50%, preferably 60-75%, more preferably from 80-95% and, most preferably, from 98-100% pure.
  • engineered refers to an amino acid sequence that is altered with respect to a natural amino acid sequence and particularly with respect to amino acids which contribute to modification-dependent binding of the polypeptide to a binding partner.
  • Naturally-occurring refers to the fact that the polypeptide or polynucleotide can be found in nature.
  • One such example is a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature.
  • the polypeptide is engineered as described herein so as to associate with a binding partner in a modification-dependent manner where it did not formerly do so or where it did so in a manner different, either in degree or kind, from that which it was engineered to do, it is no longer naturally-ocurring but is derived from a naturally ocurring polypeptide.
  • post-translational modification is reversible, such that a repeating cycles of addition and removal of a modifying moiety may be observed, although such cycles may not occur in a living cell found in nature.
  • the term “associates” or “binds” refers to a polypeptide as described herein and its binding partner having a binding constant sufficiently strong to allow detection of binding by fluorescent or other detection means, which are in physical contact with each other and have a dissociation constant (Kd) of about lO ⁇ M or lower.
  • the contact region may include all or parts of the two molecules.
  • the terms “substantially dissociated” and “dissociated” or “substantially unbound” or “unbound” refer to the absence or loss of contact between such regions, such that the binding constant is reduced by an amount which produces a discernable change in a signal compared to the bound state, including a total absence or loss of contact, such that the proteins are completely separated, as well as a partial absence or loss of contact, so that the body of the proteins are no longer in close proximity to each other but may still be tethered together or otherwise loosely attached, and thus have a dissociation constant greater than lO ⁇ M (Kd). In many cases, the Kd will be in the mM range.
  • complex and, particularly, “dimer”, “multimer” and “oligomer”as used herein, refer to the engineered binding domain and its binding partner in the associated or bound state. More than one molecule of each of engineered binding domain and its binding partner may be present in a complex, dimer, multimer or oligomer according to the methods of the invention.
  • binding sequence refers to that portion of a polypeptide comprising at least 1, but also 2 or more, preferably 4 or more, and up to 8, 10, 100 or 1000 contiguous (i.e., covalently linked by peptide bonds) amino acid residues or even as many contiguous residues as are comprised by a full-length protein, that are sufficient for modification-dependent binding to a binding partner.
  • a binding sequence may exist on a polypeptide molecule that consists solely of binding sequence amino acid residues or may, instead, be found in the context of a larger polypeptide chain (i.e., one that comprises amino acids other than those of the binding sequence).
  • engineered binding sequence refers to a binding sequence, as defined above, that is altered (e.g., by insertion, deletion or substitution of at least one amino acid) such that the fragment amino acid sequence is no longer as found in nature.
  • alteration must occur in those amino acids of a polypeptide which contribute to modification-state-dependent binding (that is, within the binding domain).
  • binding polypeptide refers to a molecule comprising multiple binding sequences, as defined above, which sequences are derived from a single, naturally-occurring polypeptide molecule and are both necessary and, in combination, sufficient to permit modification-state-dependent binding of the binding polypeptide to its binding partner, as defined above, wherein the sequences of the binding polypeptide are either contiguous or are non-contiguous.
  • non-contiguous refers to binding sequences which are linked by intervening naturally-occurring, as defined herein, or non-natural amino acid sequences or other chemical or biological linker molecules such are known in the art.
  • the amino acids of a polypeptide that do not significantly contribute to the modification- state-dependent binding of that polypeptide to its binding partner may be those amino acids which are naturally present and link the binding sequences in a binding polypeptide or they may be derived from a different natural polypeptide or may be wholly non-natural.
  • a binding polypeptide and its binding partner (which may, itself, be a binding domain, sequence or polypeptide, as defined herein) may exist on two different polypeptide chains or on a single polypeptide chain.
  • engineered binding polypeptide refers to a binding polypeptide. as defined above, which polypeptide comprises at least one engineered binding sequence, as described above.
  • a naturally-occurring amino acid sequence which links binding fragments in a binding polypeptide of use in an assay of the invention may be derived from the same natural polypeptide sequence from which one or more of the component binding fragments are drawn, including that from which an engineered binding fragment may have been derived, or may instead be derived from a different natural polypeptide.
  • the site comprises a sequence which directs modification by one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP- ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
  • a kinase e.g., a phosphatase, a
  • the site permits addition of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the addition prevents binding of the isolated engineered binding domain to the binding partner.
  • a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the addition prevents binding of the isolated engineered binding domain to the binding partner.
  • prevents binding or “prevents association” refers to the ability of at least one of the following: phosphate, ubiquitin, glycosyl, fatty acyl, sentrin or ADP-ribosyl group to inhibit the association, as defined above, of an isolated engineered binding domain and a binding partner thereof by at least 10%, preferably by 25-50%), highly preferably by 75-90%) and, most preferably, by 95-100% relative the association observed in the absence of such a modification under the same experimental conditions.
  • the site permits addition of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP- ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the addition promotes binding of the isolated engineered binding domain to the binding partner.
  • a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP- ribosyl moiety, a fatty acyl moiety and a sentrin moiety
  • promotes binding refers to that which causes an increase in binding of the engineered binding domain and its binding partner of at least two-fold, preferably 10- to 20-fold, highly preferably 50- to 100-fold, more preferably from 200- to 1000-fold, and, most preferably, from 200 to 10,000-fold.
  • the site permits removal of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the removal prevents binding of the isolated engineered binding domain to the binding partner.
  • a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety
  • the site permits removal of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the removal promotes binding of the isolated engineered binding domain to the binding partner.
  • a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety
  • At least one of the isolated engineered binding domain and the binding partner comprises a detectable label, more preferred that the detectable label emits light and most preferred that the light is fluorescent.
  • fluorescent tag refers to either a fluorophore or a fluorescent protein or fluorescent fragment thereof.
  • fluorescent protein refers to any protein which fluoresces when excited with appropriate electromagnetic radiation. This includes proteins whose amino acid sequences are either natural or engineered.
  • a “fluorescent protein” is a full-length fluorescent protein or fluorescent fragment thereof .
  • linker refers to that which is coupled to both the donor and acceptor protein molecules, such as an amino acid sequence joining two engineered binding domains, sequences or polypeptides or joining an engineered binding domain, sequence or polypeptide and its corresponding binding partner, or a disulfide bond between two polypeptide sequences, whether the sequences are present on the same- or on different polypeptide chains.
  • the reporter labels are chosen such that the emission wavelength spectrum of one (the "donor") is within the excitation wavelength spectrum of the other (the "acceptor”).
  • the fluorophore and quencher are chosen such that the emission wavelength spectrum of the fluorophore is within the absorption spectrum of the quencher such that when the fluorophore and the quencher with which it is employed are brought into close proximity by binding of the engineered binding domain, sequence or polypeptide upon which one is present with the binding partner comprising the other, detection of the fluorescent signal emitted by the fluorophore is reduced by at least 10%, preferably 20-50%, more preferably 70-90% and. most preferably, by 95-100%.
  • a typical quencher reduces detection of a fluorescent signal by approximately 80%>.
  • one of the isolated engineered binding domain and the binding partner comprises a quencher for the detectable label.
  • the invention additionally provides a kit comprising an isolated engineered binding domain and a binding partner therefor, wherein the isolated engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site, and packaging materials therefor.
  • the kit further comprises a buffer which permits modification- dependent binding of the isolated engineered binding domain and the binding partner.
  • buffer refers to a medium which permits activity of the protein-modifying enzyme used in an assay of the invention, and is typically a low-ionic- strength buffer or other biocompatible solution (e.g., water, containing one or more of physiological salt, such as simple saline, and/or a weak buffer, such as Tris or phosphate, or others as described hereinbelow), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate.
  • Such a buffer permits dimerization of a non- phosphorylated and/or non-ubiquitinated and/or non-prenylated and/or non-sentrinated and/or non-ADP-ribosylated and/or non-glycosylated engineered binding domain of the invention and a binding partner therefor and, preferably, inhibits degradation and maintains biological activity of the reaction components.
  • Inhibitors of degradation such as protease inhibitors (e.g., pepstatin, leupeptin, etc.) and nuclease inhibitors (e.g., DEPC) are well known in the art.
  • an appropriate buffer may comprise a stabilizing substance such as glycerol, sucrose or polyethylene glycol.
  • physiological buffer refers to a liquid medium that mimics the salt balance and pH of the cytoplasm of a cell or of the extracellular milieu, such that post-translational protein modification reactions and protei protein binding are permitted to occur in the buffer as they would in vivo.
  • the buffer additionally permits modification of the site for protein modification by one or more of the following enzymes: a kinase.
  • a phosphatase a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
  • carbohydrate transferase e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotrans
  • the kit further comprises one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine- Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
  • a kinase e.g., a phosphatase, a carbohydrate transferase (
  • kit further comprises a substrate for said enzyme which may be: MgATP, ubiquitin, sentrin, nicotinamide adenine dinucleotide (NAD + ), uridine-diphosphate-N-acetylglucosamine-dolichyl-phosphate (UDP-N-acetylglucosamine- dolichyl-phosphate), palmytyl CoA, myristoyl CoA and UDP-N-acetylglucosamine.
  • a substrate for said enzyme which may be: MgATP, ubiquitin, sentrin, nicotinamide adenine dinucleotide (NAD + ), uridine-diphosphate-N-acetylglucosamine-dolichyl-phosphate (UDP-N-acetylglucosamine- dolichyl-phosphate), palmytyl CoA, myristoyl CoA and UDP-N-acetylglucosamine
  • At least a part of a substrate of an enzyme of use in the invention is transferred to an modification site on an isolated engineered binding domain of the invention.
  • the term "at least a part of a substrate” refers to a portion (e.g., a fragment of an amino acid sequence, a moiety or a group, as defined above) which comprises less than the whole of the substrate for the enzyme, the transfer of which portion to a modification site on an isolated engineered binding domain and, optionally, to a site on a binding partner therefor, both as defined above, is catalyzed by the enzyme.
  • the kit further comprises a cofactor for said enzyme.
  • Cofactors of use in the invention include, but are not limited to, cAMP, phosphotidylserine, diolein, Mn 2+ and Mg 2+ .
  • At least one of the isolated engineered binding domain and the binding partner comprises a detectable label, more preferred that the detectable label emits light and most preferred that the light is fluorescent.
  • An enzyme of use in the invention may be natural or recombinant or, alternatively, may be chemically synthesized. If either natural or recombinant, it may be substantially pure
  • partially purified i.e.. represented by at least 1% of the molecules present in a fraction of a cellular lysate
  • sample refers to a collection of inorganic, organic or biochemical molecules which is either found in nature (e.g., in a biological- or other specimen) or in an artificially-constructed grouping, such as agents which might be found and/or mixed in a laboratory. Such a sample may be either heterogeneous or homogeneous.
  • biological specimen and “biological sample” refer to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • Bio sample further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
  • biological sample refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
  • organism refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
  • the detection step is to detect a change in signal emission by the detectable label.
  • the method further comprises exciting the detectable label and monitoring fluorescence emission.
  • the enzyme is one of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
  • a kinase e.g., a phosphatase, a carbohydrate transferase (e.
  • the method further comprises the step, prior to or after the detection step, of contacting the isolated engineered binding domain and the binding partner with an agent which modulates the activity of the enzyme.
  • modulate refers to enhancing or inhibiting the activity of a protein-modifying enzyme in an assay of the invention; such modulation may be direct (e.g. including, but not limited to, cleavage of- or competitive binding of another substance to the enzyme) or indirect (e.g. by blocking the initial production or, if required, activation of the modifying enzyme).
  • Modulation refers to the capacity to either increase or decease a measurable functional property of biological activity or process (e.g., enzyme activity or receptor binding) by at least 10%, 15%, 20%>, 25%, 50%, 100%o or more; such increase or decrease may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
  • a measurable functional property of biological activity or process e.g., enzyme activity or receptor binding
  • modulator refers to a chemical compound (naturally occurring or non- naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non- peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
  • Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein.
  • the activities (or activity) of a modulator may be known, unknown or partially-known. Such modulators can be screened using the methods described herein.
  • test modulator refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator. Usually, various predetermined concentrations are used for screening such as 0.01 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, and 10.0 ⁇ M, as described more fully hereinbelow.
  • Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
  • the invention also provides a method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El.
  • a kinase e.g., a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase)
  • a ubiquitin activating enzyme e.g., a ubiquitin activating enzyme
  • a ubiquitin conjugating enzyme E2 a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase
  • the method comprising contacting an isolated engineered binding domain, a binding partner therefor and an enzyme with a candidate modulator of the enzyme, wherein the engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner that is dependent upon modification of the site by the enzyme, and wherein at least one of the isolated engineered binding domain and the binding partner comprises a detectable label, and monitoring the binding of the isolated engineered binding domain to the binding partner, wherein binding or dissociation of the isolated engineered binding domain and the binding partner as a result of the contacting is indicative of modul
  • the detectable label emits light and highly preferred that the light is fluorescent.
  • the monitoring comprises measuring a change in energy transfer between a label present on the isolated engineered binding domain and a label present on the binding partner.
  • the invention also provides a method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase, the method comprising contacting an assay system with a candidate
  • the method comprises real-time observation of association of an isolated engineered binding domain and its binding partner.
  • the term "real-time” refers to that which is performed contemporaneously with the monitored, measured or observed events and which yields a result of the monitoring, measurement or observation to one who performs it simultaneously, or effectively so, with the occurrence of a monitored, measured or observed event.
  • a "real time” assay or measurement contains not only the measured and quantitated result, such as fluorescence, but expresses this in real time, that is, in hours, minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shorter times exceed the instrumentation capability; further, resolution is also limited by the folding and binding kinetics of polypeptides.
  • Figure 1 depicts double- and single-chain enzymatic assay formats of the invention.
  • Figure 2 presents a schematic overview of FRET in an assay of the invention.
  • Figure 3 presents monomer: excimer fluorescence.
  • Figure 4 presents sUb-N protein and sUb-C phage interaction monitored by surface plasmon resonance (SPR).
  • Figure 5 presents the detection of bound ZAPGFP protein by GFP fluorescence.
  • the invention is based upon the discovery that a binding polypeptide or a polypeptide comprising a binding domain or sequence, each as defined herein, which binding polypeptide, domain or sequence has been engineered so as to associate with a binding partner in a manner that is dependent upon the presence or absence of a "moiety", as described herein, at a site for post-translational modification on the same polypeptide chain provides a sensitive system for the activity of an enzyme that catalyzes post-translational protein modification at such a site and that measurements of enzymatic activity performed in such a system may be taken in real time.
  • An assay of the invention utilizes at least one polypeptide chain which comprises a sequence that has been engineered to associate specifically with a second sequence, or "binding partner" as defined herein, in a modification-dependent manner.
  • Mono-ADP-ribosylation is a post-translational modification of proteins which is currently thought to play a fundamental role in cellular signalling.
  • a number of mono-ADP- ribosyl-transferases have been identified, including endogenous enzymes from both bacterial and eukaryotic sources and bacterial toxins.
  • a mono-ADP-riboylating enzyme using as substrates the protein to be modified and nicotinamide adenine dinucleotide (NAD + ), is NAD:Arginine ADP ribosyltransferase (Zolkiewska et al, 1992, Proc. Natl. Acad. Sci. U.S.A., 89: 11352-11356).
  • This toxin induces the mono-ADP-ribosylation of BARS-50 (a G protein involved in membrane transport) and glyceraldehyde-3 -phosphate dehydrogenase.
  • the cellular effects of brefeldin A include the blocking of constitutive protein secretion and the extensive disruption of the Golgi apparatus.
  • Inhibitors of the brefeldin A mono-ADP-ribosyl- transferase reaction have been shown to antagonise the disassembly of the Golgi apparatus induced by the toxin (Weigert et al., 1997. J. Biol. Chem., 272: 14200-14207).
  • a number of amino acid residues within proteins have been shown to function as ADP-ribose acceptors.
  • Bacterial transferases have been identified which modify arginine, asparagine, cysteine and diphthamide residues in target proteins.
  • Endogenous eukaryotic transferases are known which also modify these amino acids, in addition there is evidence that serine, threonine, tyrosine, hydroxyproline and histidine residues may act as ADP-ribose acceptors but the relevant transferases have not yet been identified (Cervantes-Laurean et al., 1997, Methods Enzvmol., 280: 275-287 and references therein).
  • Poly-ADP-ribosylation is thought to play an important role in events such as DNA repair, replication, recombination and packaging and also in chromosome decondensation.
  • the enzyme responsible for the poly-ADP-ribosylation of proteins involved in these processes is poly (ADP-ribose) polymerase (PARP; for Drosophila melanogaster PARP, see Genbank Accession Nos. D 13806, D 13807 and D 13808).
  • PARP poly (ADP-ribose) polymerase
  • ADP ADP-ribosylation sites are those found at Cys 3 and Cys 4 (underlined) of the B-50 protein (Coggins et al., 1993, J. Neurochem.. 60: 368-371; SwissProt Accession No. P06836): MLCCMRRTKQVE KNDDD and P ⁇ (the ⁇ subunit of cycylic CMP phophodiesterase; Bondarenko et al., 1997, J. Biol. Chem.. 272: 15856-15864; Genbank Accession No. X04270): FKQRQTRQFK .
  • Ubiquitination of a protein targets the protein for destruction by the proteosome. This process of destruction is very rapid (X. ⁇ ⁇ 60 seconds), and many proteins with rapid turnover kinetics are destroyed via this route. These include cyclins, p53, transcription factors and transcription regulatory factors, among others. Thus, ubiquitination is important in processes such as cell cycle control, cell growth, inflammation, signal transduction; in addition, failure to ubiquitinate proteins in an appropriate manner is implicated in malignant transformation.
  • Ubiquitin is a 76-amino-acid protein which is covalently attached to a target protein by an isopeptide bond, between the ⁇ -amino group of a lysine residue and the C-terminal glycine residue of ubiquitin. Such modification is known as mono-ubiquitination, and this can occur on multiple Lys residues within a target protein.
  • the ubiquitin can itself be ubiquitinated, thus forming extended branched chains of polyubiquitin. It is this latter state which signals destruction of the target protein by the proteosome. In the process of destruction, it appears that the polyubiquitinated protein is taken to the proteosome via a molecular chaperone protein, the ubiquitin molecules are removed undamaged (and recycled) and the target is degraded.
  • Ubiquitination is a complex process, which requires the action of three enzymes: Ubiquitin activating enzyme El (for human, Genbank Accession No. X56976), ubiquitin conjugating enzyme E2, also referred to as the ubiquitin carrier protein, (for human 17kDa form, Genbank Accession No. X78140) and Ubiquitin protein ligase E3 ⁇ (UBR1; human, Genbank Accession No. AF061556).
  • El for human, Genbank Accession No. X56976
  • E2 also referred to as the ubiquitin carrier protein
  • Ubiquitin protein ligase E3 ⁇ Ubiquitin protein ligase E3 ⁇
  • the signals contained within a protein which determine whether the protein is subject to the process of ubiquitination and destruction are two-fold: first, the identity of the N- terminal amino acid (so called N-end rule, Varshavsky, 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 12142-12149), and secondly the presence of a suitably positioned Lys residue in the protein (Varshavsky, 1996, supra).
  • This Lys can be up to -30 amino acids away from the N- terminus in experimental examples studied where the N-terminus is a flexible, poorly- structured element of the protein (Varshavsky, 1996, supra) or could potentially be anywhere in the sequence where this presents it at an appropriate location relative to the N-terminus.
  • N-terminal residues can be classed as stabilizing (s) or destabilizing (d), and the inclusion of an amino acid in one of these broad classes is species-dependent (prokaryotes differ from yeast, which differs from mammals; Varshavsky, 1996, supra).
  • the destabilizing N-terminal residue and the internal Lys can be in cis (on a single peptide), but may also be in trans (on two different polypeptides). The tr ⁇ r ⁇ '-recognition event will only take place while the complex is physically associated. Only the ubiquitinated subunit is proteolyzed (Varsharsky, 1996, supra).
  • ubiquitinated lysine residue is underlined for each (e.g., Lys* 5 and Lys ⁇ for ⁇ - galactosidase).
  • a ubiquitination assay measures the addition of ubiquitin to-, rather than the destruction of-, an engineered binding domain, sequence or polyeptpide.
  • Glycosylation N-linked glycosylation is a post-translational modification of proteins which occurs in the endoplasmic reticulum and golgi apparatus and is utilized with some proteins en route for secretion or destined for expression on the cell surface or in another organelle.
  • the carbohydrate moiety is attached to Asn residues in the non-cytoplasmic domains of the target proteins, and the consensus sequence (Shakineshleman, 1996, Trends Glvcosci. Glycotech., 8: 115-130) for a glycosylation site is: NxS/T, where x cannot be proline or aspartic acid.
  • N-linked sugars have a common five-residue core consisting of two GlcNAc residues and three mannose residues due to the biosynthetic pathway.
  • This core is modified by a variety of Golgi enzymes to give three general classes of carbohydrate known as oligomarmosyl, hybrid and lactosamine-containing or complex structures (Zubay, 1998, Biochemistry, Wm. C. Brown Publishers).
  • Oxygen-linked glycosylation also occurs in nature with the attachment of various sugar moieties to Ser or Thr residues (Hansen et al., 1995, Biochem. J.. 308: 801-813).
  • Intracellular proteins are among the targets for O-glycosylation through the dynamic attachment and removal of O-N-Acetyl-D-glucosamine (O-GlcNAc; reviewed by Hart, 1997, Ann. Rev. Biochem., 66: 315-335).
  • Proteins known to be O-glycosylated include cytoskeletal proteins, transcription factors, the nuclear pore protein complex, and tumor- suppressor proteins (Hart, 1997, supra).
  • O-GlcNAc specific sites for the addition of O-GlcNAc are found, for example, at Ser 277 , Ser 3 ⁇ 6 and Ser 383 of p67 SRF (Reason et al., 1992, J. Biol. Chem., 267: 16911-16921 ; Genbank Accession No. J03161). The recognition sequences encompassing these residues are shown below: 74 GTTSTIQTAP 313 SAVSSADGTVLK 374 DSSTDLTQTSSSGTVTLP The identity of sites of O-GlcNAc is additionally known for a small number of proteins including c-myc (Thr 58 , also a phosphorylation site; Chou et al., 1995, J. Biol. Chem., 270: 18961-18965), the nucleopore protein p62 (see Reason et al., 1992, supra):
  • the post-translational modification of proteins with fatty acids includes the attachment of myristic acid to the primary amino group of an N-terminal glycine residue (Johnson et al., 1994, Ann. Rev. Biochem., 63: 869-914) and the attachment of palmitic acid to cysteine residues (Milligan et al., 1995. Trends Biochem. Sci., 20: 181-186).
  • Fatty acylation of proteins is a dynamic post-translational modification which is critical for the biological activity of many proteins, as well as their interactions with other proteins and with membranes.
  • the location of the protein within a cell can be controlled by its state of prenvlation (fatty acid modification) as can its ability to interact with effector enzymes (ras and MAP kinase, Itoh et al., 1993, J. Biol. Chem., 268: 3025-; ras and adenylate cyclase (in yeast; Horiuchi et al., 1992, Mol. Cell. Biol., 12: 4515-) or with regulatory proteins (Shirataki et al., 1991, J.
  • Sentrin is a novel 101-amino acid protein which has 18 % identity and 48% similarity with human ubiquitin (Okura et al., 1996, J. Immunol., 157: 4277-4281). This protein is known by a number of other names including SUMO- 1 , UBL 1 , PIC 1 , GMP 1 and SMT3 C and is one of a number of ubiquitin-like proteins that have recently been identified. Sentrin is expressed in all tissues (as shown by Northern blot analysis), but mRNA levels are higher in the heart, skeletal muscle, testis, ovary and thymus.
  • RanGAPl Ran-specific GTPase-activating protein
  • NPC nuclear pore complex
  • Fas/APOl and TNF receptors are involved in transducing the apoptosis signal via their death domains. Ligation of Fas on the cell surface results in the formation of a complex via death domains and death-effector domains, triggering the induction of apoptosis.
  • sentrin protects cells from both anti-Fas/ APO and TNF-induced cell death (Okura et al., 1996, supra). It is not clear whether this protection is achieved simply by preventing the binding of other proteins to these death domains or whether a more complex process is involved, possibly one involving the ubiquitin pathway.
  • PML a RING finger protein
  • protein kinases identified to date include the protein tyrosine kinase subfamily (such as PDGF receptors, EGF receptors, src family kinases (see Brown and Cooper, 1996, Biochimica and Biophysica Acta 1287: 121-149 for a review), the JAK kinase family (such as JAKl , JAK2 and tyk2), Erb B2, Bcr-Abl, Alk, Trk, Res/Sky - for a detailed review see Al-Obeidi et al, 1998, Biopolymers (Peptide Science), Vol 47: 197-223), the MAP kinase pathway subfamily (such as the MAP family, the ERK family, the MEK family, the MEKK family, RAF-1 and JNK), the cyclin-dependent kinase subfamily (such as p34 c c and cdk2 - see Nigg, 1995, Bioessays 17: 471-480 for
  • PK-C protein kinase C
  • PK-A cyclic-AMP dependent kinase
  • Ca2+/calmodulin dependent kinases such as CaM kinase I, II and IV
  • DNA dependent protein kinase DNA dependent protein kinase
  • PAKs p21 -activated protein kinase family
  • kinases whose activity may be studied using the methods of the invention include the src family tyrosine kinases Lck and Fyn, that phosphorylate the TCR ⁇ chain, and are known to be involved in signal transduction associated with T cell receptor stimulation.
  • the TCR ⁇ chain comprises specific tyrosine residues present in immunoreceptor tyrosine-based activation motifs (ITAMs) that are phosphorylatd by Lck and Fyn (Kuriyan and Cowburn, 1997, ibid.).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • TCR ⁇ ITAM and ZAP70 SH2 represent binding domains and binding partners that may be of interest in studying the activity of the kinases Lck and Fyn (see Elder et al., 1994, Science 264: 1596-1599 and Chan et al., 1994, Science 264: 1599- 1601.
  • Another example is the IgE receptor ⁇ subunit and the SH2 domain of Syk that may be used to study the activity of the Lyn kinase.
  • PPP family includes the following catalytic subunits: PPlc, PP2Ac, PP2B, PPP1, PPP2A and PPP5 and the following regulatory subunits: NIPP-1, RIPP-1, p53BP2, ⁇ ,34.5, PR65, PR55, PR72, PTPA, SV40 small T antigen, PPY, PP4, PP6 and PP5.
  • the PPM family includes pyruvate dehydrogenase phosphatase and Arabidopsis ABU .
  • the protein tyrosine phosphatase family includes PTP1B, SHP-1, SHP-2 (cytosolic non- receptor forms), CD45 (see Thomas and Brown, 1999, Trends in Immunol, 20: 406 and Ashwell and D'Oro, 1999, Trends in Immunol, 20: 412 for further details), RPTP (receptorlike, transmembrane forms) and cdc25, kinase-associated phosphatase and MAP kinase phosphatase- 1 (dual-specificity phosphatases).
  • PTP1B is known to associate with the insulin receptor in vivo (Bandyopadhyay et al., 1997, J. Biol. Chem. 272: 1639-1645).
  • an assay of the invention may be performed using either a single-chain or double- chain format, as illustrated in Figure 1.
  • an engineered binding domain may associate with a second amino acid sequence (or "binding partner") present on the same polypeptide chain or, alternatively, with a binding partner present on a second polyepeptide chain.
  • the binding partner may, itself, be an engineered binding polypeptide (i.e., one which is engineered so as to be dependent upon the presence or absence of a chemical moiety at a site for post-translational modification in order to participate in proteimprotein binding) or may be a natural sequence or a non-natural amino acid sequence which does not comprise a site for post-translational modification that affects proteimprotein binding.
  • the complex between an engineered binding domain and a binding partner may, therefore, comprise a self- associated polypeptide monomer or. alternatively, either a hetero- or homo-oligomer.
  • Non-limiting examples of pairs of amino acid sequences which associate in nature and can be engineered to provide engineered binding domains, sequences or polypeptides of use in the invention are presented in Table 1.
  • One or, alternatively, both members of a pair can be engineered to comprise sites for post-translational modification.
  • association of these two regions will have a number of determinants other than the engineered chemical modification in vivo including the presence or absence of PKC activators (such as phosphatidylserine, Ca 2+ and diacylglycerol) or endogenous RACK1.
  • PKC activators such as phosphatidylserine, Ca 2+ and diacylglycerol
  • endogenous RACK1 endogenous RACK1.
  • the SH2 domain In order for the SH2 domain to be useful in an assay of this type it must be modified such that the addition or removal of a phosphate group from a tyrosine residue is no longer a determinant of binding. This could be achieved by thiophosphorylation of the Tyr residue in an in vitro assay to yield a permanently phosphorylated protein. Alternatively, it may be possible to mimic phosphorylation by the mutuation of the key Tyr residue to Glu or Asp. If this were possible then these domains could be used in an in vivo assay. ** Wang et al... 1997, J. Biol. Chem.. 272: 17542-17550.
  • ZIP contains more than one protein binding motif (YXDED motif, ZZ zinc finger) and is known to bind to several proteins other than PDC ⁇ (including p62 and EBIAP) and also to self-associate (this self association is in competition with PKC ⁇ binding). These multiple interactions may cause problems with an in vivo assay as it is not clear whether any of the proteins bind in a competitive manner.
  • Sites for post-translational modification may be selected according to the specificity of enzyme(s) to be assayed.
  • Non-limiting examples of sites for post-translational modification are presented in Tables 2 (phosphorylation/dephosphorylation) and 3 (addition/removal of other chemical moieties).
  • X signifies any amino acid.
  • Consensus sequences are taken from Trends Biochem. Sci. (1990) 15: 342-346. Further examples are tabulated in Pearson and Kemp, 1991. Methods Enzvmol., 200: 62-81.
  • E engineered phosphorylation site
  • F2 acceptor fluorophore
  • FRET Fluorescent resonance energy transfer
  • a simple FRET assay based upon these modifications to site for post-translational modification present on an engineered binding domain, sequence or polypeptide may be performed as presented below. It is contemplated that other light-based detection assays, such as those involving single labels, labels and corresponding quenchers, etc. can be employed.
  • a FRET-based assay may follow a format such as:
  • Placement of the modification site may be determined empirically (see below), such that the location itself permits the interaction between the engineered binding domain, sequence or polypeptide and a binding partner but that the association is altered on modification of the engineered site.
  • This change in association may be a direct or indirect consequence of modification. While not being bound to any theory, such a change may be based on, for example, a conformational or electrostatic change brought about by phosphorylation or dephosphorylation. In cases where there is no appropriate structural information, the sites for the attachment of a fluorophore or other label or quencher will also be determined empirically.
  • Table 4 lists enzymes which perform the several modifications discussed herein as being of use in the invention.
  • a binding domain is engineered such that its association with a binding partner is dependent upon post-translational modification at a site for post- translational modification which is introduced into- or altered within- the binding domain.
  • the binding domain which undergoes engineering may, itself, be a naturally-occurring amino acid sequence or may be non-natural. Such engineering is performed by molecular methods which are well known in the art, as described below.
  • the location of the engineered post- translational modification site must be such that it is tolerated in one state of modification (for example, prior to modification), but provokes dissociation of the complex in the opposite state of modification (following modification; or vice versa).
  • placement of the modification site within the domain and, optionally, the corresponding binding partner may involve empirical testing on a case-by-case basis; however, such testing can be facilitated through the use of knowledge of the structural basis of the interaction sites in the complex.
  • knowledge may be structural (e.g., using crystallographic data or a molecular modeling algorithm of the 3 -dimensional structure of the protein or proteins involved in the complex of interest), a functional assessment of the regions of primary sequence important in binding, or a combination of these. These data will identify regions of the protein most likely to be influenced by the insertion of a post-translational modification site.
  • the contact face between components of the complex is one location at which a site for post-translational modification might be engineered, but it is not the only useful location.
  • the modification of a site remote from the interface site(s) can also lead to binding or dissociation of the complex. This would be expected to occur upon long-range alterations in protein structure as a consequence of the post-translational modification, which could be as extreme, for example, as structural collapse following modification.
  • a peptide, PKI(5-24amide), derived from a protein inhibitor of the cAMP-dependent protein kinase binds to the active site of protein kinase A (PKA) with high affinity.
  • PKA protein kinase A
  • the 3-D structure of this complex is known (Knighton et al., 1991, Science, 253: 414-420) as is a functional dissection of the sequence of this peptide to identify residues involved in this biological activity (Glass et al., 1989, J. Biol. Chem., 264: 8802-8810).
  • the binding of PKI(5-24amide) to the catalytic subunit of PKA can be monitored by a number of techniques including FRET, fluorescence correlation spectroscopy (FCS) or fluorescence anisotropy provided both components in the former case or the PKI(5-24amide) component in the latter two cases, respectively, are labeled with appropriate fluorophores.
  • FRET fluorescence correlation spectroscopy
  • FCS fluorescence correlation spectroscopy
  • PKI(A21S) results in a reporter molecule for protein kinase A activity.
  • PKI(A21S) binds to PKA when dephosphorylated, but dissociates from the enzyme once phosphorylated.
  • the binding partner might require mutation of its primary sequence to accommodate the post-translational modification site introduced into the engineered binding domain.
  • An engineered binding domain of use in the invention is produced using molecular methods such as are known in the art (see, for example, Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al, Current Protocols in Molecular Biology, copyright 1987- 1994, Current Protocols, copyright 1994-1998, John Wiley & Sons, Inc.).
  • Such methods include chemical synthesis of a polyeptide sequence that encompasses an engineered binding domain or expression of a recombinant polynucleotide encoding such a molecule.
  • Such a polynucleotide may be chemically synthesized; however, of particular use in the invention are methods of in vitro or otherwise site-directed mutagenesis by which to engineer a site for post-translational modification into an existing binding domain (whether natural or previously engineered) or by which to alter the enzyme specificity of an existing site.
  • methods for in vitro mutagenesis comprise the annealing of a mutagenic oligonucleotide primer comprising the desired alteration to a complementary, single-stranded template, followed by second strand synthesis, whether using single-cycle synthesis or polymerase chain reaction (PCR). Cloning and sequencing are then performed to identify and isolate molecules bearing the desired alterations.
  • Such mutagenesis methods optionally include a selection for mutated molecules, either through the use of modified nucleotides incorporated into the nascent polynucleotide strand or through the incorporation of a restriction site into the vector bearing the first strand which is disrupted in the second strand (i.e., in coupled priming; Carter et al., 1985, Nucleic Acids Res., 13: 4431-4443) and, with either technique, subsequent transformation of the first and second strands into a strain of host cells that selectively destroys the first strand and propagates the second.
  • Kits and individual components for in vitro mutagenesis enjoy wide commercial availability.
  • a non-limiting sampling of such kits is as follows: From Stratagene (LaJolla, CA, U.S.A.): ExSiteTM PCR-Based Site-Directed Mutagenesis Kit (catalog number: 200502)
  • Erase-a-Base® System (catalog numbers E5850 and E 5750) From New England Biolabs (Beverly, MA, U.S.A.):
  • candidate binding partners can be screened for their ability to bind the engineered binding domain in a modification-dependent manner.
  • binding partners may be selected or designed based upon sequence homology with known binding partners or on molecular modeling data (e.g., from a modeling algorithm).
  • Potential binding partners additionally may be purified (e.g, using the modified engineered binding domain as the trap in affinity chromatgraphy or as a probe for a library) from a population of polypeptide molecules.
  • a library from which to draw a diverse population of polypeptide sequences of use in the invention includes, but is not limited to, an expression library or a synthetic peptide library (see “Candidate modulators", below).
  • One library-based technique which is useful in the invention to generate new pairs of assay components is that of phage display, which provides convenient testing of polypeptide sequences able to complex with the target sequence from a vast repertoire of different polypeptide sequences.
  • Filamentous bacteriophage display a small number of copies of a protein termed g3p on their surface.
  • This protein is responsible for interacting with proteins on the surface of Escherichia coli and facilitates the infection of the bacterium.
  • This protein possesses three globular domains linked by protease resistant, flexible amino acid sequences.
  • the g3p protein can be modified to provide a means of presenting protein structures from which proteins capable of forming a stable binding complex can be identified.
  • Such a bioassay can be configured in a number of ways including: a) Expression of the test proteins as an extension of the g3p sequence. Proteins able to bind with target polypeptide A can be selected by affinity purification on a matrix displaying polypeptide A.
  • test protein as an extension of the g3p protein, plus independent expression from the same phage of the target protein (polypeptide A) fused to a convenient affinity tag (such as His 6 ). The binding of polypeptide A to the test protein displayed on g3p will facilitate the affinity purification of this phage particle.
  • binding partners tolerant of that engineered site can be identified.
  • a second round of selection can then be undertaken to identify the binding partners which dissociate upon post-translational modification of that site (i.e., those to which binding of the engineered binding domain is dependent upon post-translational modification).
  • the activity of a modifying enzyme is assayed by measuring the formation or destruction of proteimprotein complexes when the modifying enzyme is present with an engineered binding domain, sequence or polypeptide and its corresponding binding partner under conditions which permit modifying activity.
  • Methods which enable the detection of proteimprotein complexes i.e., methods which allow one of skill in the art to discriminate between polypeptide pairing partners which are bound and those which are unbound) are known in the art.
  • FRET Fluorescent resonance energy transfer
  • FRET fluorescent resonance energy transfer
  • Fluorescenceless energy transfer is based on the biophysical properties of fluorophores. These principles are reviewed elsewhere (Lakowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum Press, New York; Jovin and Jovin, 1989, Cell Structure and Function by Microspectrofluorometry, eds. E. Kohen and J.G. Hirschberg, Academic Press, both of which are incorporated herein by reference). Briefly, a fluorophore absorbs light energy at a characteristic wavelength. This wavelength is also known as the excitation wavelength. The energy absorbed by a flurochrome is subsequently released through various pathways, one being emission of photons to produce fluorescence.
  • the wavelength of light being emitted is known as the emission wavelength and is an inherent characteristic of a particular fluorophore.
  • Radiationless energy transfer is the quantum-mechanical process by which the energy of the excited state of one fluorophore is transferred without actual photon emission to a second fluorophore. That energy may then be subsequently released at the emission wavelength of the second fluorophore.
  • the first fluorophore is generally termed the donor (D) and has an excited state of higher energy than that of the second fluorophore, termed the acceptor (A).
  • the essential features of the process are that the emission specturm of the donor overlap with the excitation spectrum of the acceptor, and that the donor and acceptor be sufficiently close.
  • the distance over which radiationless energy transfer is effective depends on many factors including the fluorescence quantum efficiency of the donor, the extinction coefficient of the acceptor, the degree of overlap of their respective spectra, the refractive index of the medium, and the relative orientation of the transition moments of the two fluorophores.
  • the distance between D and A must be sufficiently small to allow the radiationless transfer of energy between the fluorophores.
  • FRET may be performed either in vivo or in vitro. Proteins are labeled either in vivo or in vitro by methods known in the art. According to the invention, an engineered binding domain, sequence or polypeptide and its corresponding binding partner, comprised either by the same or by different polypeptide molecules, are differentially labeled, one with a donor and the other with an acceptor label, and differences in fluorescence between a test assay, comprising a protein modifying enzyme, and a control, in which the modifying enzyme is absent, are measured using a fluorimeter or laser-scanning microscope. It will be apparent to those skilled in the art that excitation/detection means can be augmented by the incorporation of photomultiplier means to enhance detection sensitivity.
  • the differential labels may comprise either two different fluorescent labels (e.g., fluorescent proteins as described below or the fluorophores rhodamine, fluorescein, SPQ, and others as are known in the art) or a fluorescent label and a molecule known to quench its signal; differences in the proximity of the engineered binding domain, sequence or polypeptide and the binding partner with and without the protein-modifying enzyme can be gauged based upon a difference in the fluorescence spectrum or intensity observed.
  • fluorescent labels e.g., fluorescent proteins as described below or the fluorophores rhodamine, fluorescein, SPQ, and others as are known in the art
  • a fluorescent label and a molecule known to quench its signal e.g., differences in the proximity of the engineered binding domain, sequence or polypeptide and the binding partner with and without the protein-modifying enzyme can be gauged based upon a difference in the fluorescence spectrum or intensity observed.
  • a sample, whether in vitro or in vivo, assayed according to the invention therefore comprises a mixture at equilibrium comprising at least one* labeled engineered binding domain, sequence or polypeptide and its corresponding binding partner which, when disassociated from one another, fluoresce at one frequency and, when complexed together, fluoresce at another frequency or, alternatively, of molecules which either do or do not fluoresce depending upon whether or not they are associated.
  • a fluorescent label is either attached to the surface of the engineered binding domain, sequence or polypeptide or binding partner therefor or, alternatively, a fluorescent protein is fused in-frame with the engineered binding domain, sequence or polypeptide or binding partner therefor, as described below.
  • the choice of fluorescent label will be such that upon excitation with light, labeled peptides which are associated will show optimal energy transfer between fluorophores.
  • a protein modifying enzyme e.g., a phosphorylating-, a dephosphorylating-, a ubiquitinating-, ADP-ribosylating-, sentrinizing, prenylating- or glycosylating enzyme
  • a complex comprising an engineered binding domains, sequence or polypeptides and its binding partner dissociates due to structural or electrostatic disruption which occurs as a consequence of modification of the enzyme recognition site, thereby leading to a decrease in energy transfer and increased emission of light by the donor fluorophore.
  • a protein modifying enzyme e.g., a phosphorylating-, a dephosphorylating-, a ubiquitinating-, ADP-ribosylating-, sentrinizing, prenylating- or glycosylating enzyme
  • a complex comprising an engineered binding domains, sequence or polypeptides and its binding partner dissociates due to structural or electrostatic disruption which occurs as a consequence of modification of the enzyme recognition site,
  • fluorophore and “fluorochrome” refer interchangeably to a molecule which is capable of absorbing energy at a wavelength range and releasing energy at a wavelength range other than the absorbance range.
  • excitation wavelength refers to the range of wavelengths at which a fluorophore absorbs energy.
  • emission wavelength refers to the range of wavelength that the fluorophore releases energy or fluoresces.
  • fluorescent proteins which vary among themselves in excitation and emission maxima are listed in Table 1 of WO 97/28261 (Tsien et al., 1997, supra). These (each followed by [excitation max./emission max.] wavelengths expressed in nanometers) include wild-type Green Fluorescent Protein [395(475)/508] and the cloned mutant of Green Fluorescent Protein variants P4 [383/447], P4-3 [381/445], W7 [433(453)/475(501)], W2 [432(453)/480], S65T [489/511], P4-1 [504(396)/480], S65A [471/504], S65C [479/507], S65L [484/510], Y66F [360/442], Y66W [458/480], I0c [513/527], W1B [432(453)/476(503)], Emerald [487/508] and Sapphire [395/511].
  • a one embodiment of the technology can utilize monomer:excimer fluorescence as the output.
  • monomer:excimer fluorescence as the output.
  • the association of an engineered binding domains, sequence or polypeptide with a binding partner in this format is shown in Figure 3.
  • the fluorophore pyrene when present as a single copy displays fluorescent emission of a particular wavelength significantly shorter than when two copies of pyrene form a planar dimer (excimer), as depicted.
  • excitation at a single wavelength is used to review the excimer fluorescence ( ⁇ 470nm) over monomer fluorescence ( ⁇ 375nm) to quantify assembly: disassembly of the reporter molecule.
  • FCS fluorescence correlation spectroscopy
  • FCS Fluorescence-Activated Cell Sorting
  • Each individual burst, resulting from a single molecule, can be registered.
  • a labeled polypeptide will diffuse at a slower rate if it is large than if it is small. Thus, multimerized polypeptides will display slow diffusion rates, resulting in a lower number of fluorescent bursts in any given timeframe, while labeled polypeptides which are not multimerized or which have dissociated from a multimer will diffuse more rapidly. Binding of polypeptides according to the invention can be calculated directly from the diffusion rates through the illuminated volume. Where FCS is employed, rather than FRET, it is not necessary to label more than one polypeptide. Preferably, a single polypeptide member of the multimer is labeled. The labeled polypeptide dissociates from the multimer as a result of modification, thus altering the FCS reading for the fluorescent label.
  • a further detection technique which may be employed in the method of the present invention is the measurement of time-dependent decay of fluorescence anisotropy. This is described, for example, in Lacowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum Press, New York, incorporated herein by reference (see, for example, page 167).
  • Fluorescence anisotropy relies on the measurement of the rotation of fluorescent groups. Larger multimers of polypeptides rotate more slowly than monomers, allowing the formation of multimers to be monitored.
  • the invention may be configured to exploit a number of non-fluorescent labels.
  • the engineered binding domain and binding partner therefor form, when bound, an active enzyme which is capable of participating in an enzyme-substrate reaction which has a detectable endpoint.
  • the enzyme may comprise two or more polypeptide chains or regions of a single chain, such that upon binding of the engineered binding domain to the binding partner, which are present either on two different polypeptide chains or in two different regions of a single polypeptide, these components assemble to form a functional enzyme.
  • Enzyme function may be assessed by a number of methods, including scintillation counting and photospectroscopy.
  • the invention may be configured such that the label is a redox enzyme, for example glucose oxidase, and the signal generated by the label is an electrical signal.
  • Modification of the engineered binding domain and, optionally, its binding partner according to the invention is required to inhibit binding and, consequently, enzyme component assembly, thus reducing enzyme activity.
  • an enzyme is used together with a modulator of enzyme activity, such as an inhibitor or a cofactor.
  • a modulator of enzyme activity such as an inhibitor or a cofactor.
  • one of the enzyme and the inhibitor or cofactor is an engineered binding domain, the other its binding partner. Binding of the enzyme to its inhibitor or cofactor results in modulation of enzymatic activity, which is detectable by conventional means (such as monitoring for the conversion of substrate to product for a given enzyme).
  • the fluorescent protein labels are chosen such that the excitation spectrum of one of the labels (the acceptor) overlaps with the emission spectrum of the excited fluorescent label (the donor).
  • the donor is excited by light of appropriate intensity within the donor's excitation spectrum.
  • the donor then emits some of the absorbed energy as fluorescent light and dissipates some of the energy by FRET to the acceptor fluorescent label.
  • the fluorescent energy it produces is quenched by the acceptor fluorescent label.
  • FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor, reduction in the lifetime of its excited state, and re-emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor.
  • FRET is diminished or eliminated.
  • a single polypeptide may comprises a blue fluorescent protein donor label and a green fluorescent protein acceptor label, wherein one is fused to an engineered binding domain, sequence or polypeptide and the other is fused to its corresponding binding partner within that polypeptide; such a construct is herein referred to as a "tandem" fusion protein.
  • tandem fusion proteins two distinct polypeptides, (single" fusion proteins) one comprising an engineered binding domain, sequence or polypeptide and the other its corresponding binding partner, may be differentially labeled with the donor and acceptor fluorescent protein labels, respectively.
  • tandem fusion proteins in the invention can reduce significantly the molar concentration of peptides necessary to effect an association between differentially-labeled engineered species and their respective binding partners relative to that required when single fusion proteins are instead used.
  • the labeled engineered binding domains, sequences or polypeptides and their corresponding binding partners may be produced via the expression of recombinant nucleic acid molecules comprising an in-frame fusion of sequences encoding an engineered binding domain, sequence or polypeptide or a binding partner therefor and a fluorescent protein label either in vitro (e.g., using a cell-free transcription/translation system, as described below, or instead using cultured cells transformed or transfected using methods well known in the art) or in vivo, for example in a trangenic animal including, but not limited to, insects, amphibians and mammals.
  • a recombinant nucleic acid molecule of use in the invention may be constructed and expressed by molecular methods well known in the art, and may additionally comprise sequences including, but not limited to, those which encode a tag (e.g., a histidine tag) to enable easy purification, a secretion signal, a nuclear localization signal or other primary sequence signal capable of targeting the construct to a particular cellular location, if it is so desired.
  • a tag e.g., a histidine tag
  • acceptor pairs of flurescent proteins include, but are not limited to:
  • Donor S72A, K79R, Y145F, M153A and T203I (excitation ⁇ 395nm; emission ⁇ 511)
  • Acceptor S65G, S72A, K79R and T203Y (excitation ⁇ 514 nm; emission ⁇ 527), or T203Y/S65G, V68L, Q69K or S72A (excitation ⁇ 515nm; emission ⁇ 527nm).
  • P4-3 shown in Table 1 of Tsien et al., 1997, supra
  • S65C also of Table 1 of Tsien et al., 1997, supra
  • the mixtures comprising engineered binding domains, sequences or polypeptides and their corresponding binding partners are exposed to light at, for example, 368 nm, a wavelength that is near the excitation maximum of P4-3. This wavelength excites S65C only minimally.
  • some portion of the energy absorbed by the blue fluorescent protein label is transferred to the acceptor label through FRET if the engineered binding domain, sequence or polypeptide and its corresponding binding partner are in close association.
  • the acceptor label may re-emit the energy at longer wavelength, in this case, green fluorescent light.
  • modification e.g., phosphorylation, ADP-ribosylation, ubiquitination, prenylation, sentrination or glycosylation, all as described below
  • modification e.g., phosphorylation, ADP-ribosylation, ubiquitination, prenylation, sentrination or glycosylation, all as described below
  • modification e.g., phosphorylation, ADP-ribosylation, ubiquitination, prenylation, sentrination or glycosylation, all as described below
  • the two and, hence, the green and red or, less preferably, green and blue fluorescent proteins physically separate or associate, accordingly inhibiting or promoting FRET.
  • the intensity of visible blue fluorescent light emitted by the blue fluorescent protein increases, while the intensity of visible green light emitted by the green fluorescent protein as a result of FRET, decreases.
  • Such a system is useful to monitor the activity of enzymes that modify the engineered binding domain, sequence or polypeptide or binding partner to which the fluorescent protein labels are fused as well as the activity of modulators or candidate modulators of those enzymes.
  • this invention contemplates assays in which the amount- or activity of a modifying enzyme in a sample is determined by contacting the sample with an engineered binding domain, sequence or polypeptide and its binding partner, differentially labeled with fluorescent proteins, as described above, and measuring changes in fluorescence of the donor label, the acceptor label or the relative fluorescence of both.
  • Fusion proteins as described above, which comprise either one or both labeled polypeptides comprising engineered binding domains, sequences or polypeptides and the corresponding binding partner of an assay of the invention can be used for, among other things, monitoring the activity of a modifying enzyme inside the cell that expresses the recombinant tandem construct or two different recombinant constructs.
  • Single- and tandem fluorescent fusion constructs include the greater extinction coefficient and quantum yield of many of these proteins compared with those of the Edans fluorophore.
  • the acceptor in such a construct or pair of constructs is, itself, a fluorophore rather than a non-fluorescent quencher like Dabcyl.
  • the enzyme's substrate i.e., the engineered binding domain, sequence or polypeptide (and, optionally, the binding partner) comprising a post-translational modification site and product (i.e., the engineered binding domain, sequence or polypeptide and its binding partner after addition or removal of a chemical moiety to/from the modification site) are both fluorescent, but with different fluorescent characteristics.
  • the substrate and modified products exhibit different ratios between the amount of light emitted by the donor and acceptor labels. Therefore, the ratio between the two fluorescences measures the degree of conversion of substrate to products, independent of the absolute amount of either, the thickness or optical density of the sample, the brightness of the excitation lamp, the sensitivity of the detector, etc. Furthermore, Aequorea-de ⁇ ved or - related fluorescent protein labels tend to be protease resistant. Therefore, they are likely to retain their fluorescent properties throughout the course of an experiment.
  • nucleic acid constructs of particular use in the invention are those which comprise in-frame fusions of sequences encoding an engineered binding domain, sequence or polypeptide, and/or a binding partner therefor, and a fluorescent protein. If an engineered binding domain, sequence or polypeptide and its binding partner are to be expressed as part of a single polypeptide, the nucleic acid molecule additionally encodes, at a minimum, a donor fluorescent protein label fused to one, an acceptor fluorescent protein label fused to the other, a linker that couples them and is of sufficient length and flexibility to allow for folding of the polypeptide and pairing of the engineered binding domain, sequence or polypeptide and its binding partner and gene regulatory sequences operatively linked to the fusion coding sequence.
  • each nucleic acid molecule need only encode a polypeptide comprising an engineered domain, sequence or polypeptide or its binding partner fused either to a donor or acceptor fluorescent protein label and operatively linked to gene regulatory sequences.
  • control sequences refers to polynucleotide sequences which are necessary to effect the expression of coding and non-coding sequences to which they are ligated.
  • the nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • a donor fluorescent protein label is capable of absorbing a photon and transferring energy to another fluorescent label.
  • the acceptor fluorescent protein label is capable of absorbing energy and emitting a photon.
  • a fluorophore emits fluorescent light which is absorbed by a quencher.
  • the linker connects the engineered binding domain, sequence or polypeptide either directly or indirectly, through an intermediary linkage with one or both of the donor and acceptor fluorescent protein labels or the fluorescent label and, optionally, the quencher if a non-FRET assay is being performed.
  • a fluorescent protein of use in the invention includes, in addition to those with intrinsic fluorescent properties, proteins that fluoresce due intramolecular rearrangements or the addition of cofactors that promote fluorescence.
  • green fluorescent proteins of cnidarians, which act as their energy-transfer acceptors in bioluminescence, can be used in the invention.
  • a green fluorescent protein as used herein, is a protein that fluoresces green light
  • a blue fluorescent protein is a protein that fluoresces blue light.
  • GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, from the sea pansy, Renilla reniformis, and from Phialidium gregarium. (Ward et al, 1982, Photochem. PhotobioL, 35: 803-808; Levine et al. 1982. Comp. Biochem. Phvsiol..72B: 77-85).
  • a variety of Aequorea-r elated GFPs having useful excitation and emission spectra have been engineered by modifying the amino acid sequence of a naturally-occurring GFP from Aequorea victoria. (Prasher et al, 1992, Gene, 111 : 229-233; Heim et al., 1994, Proc. Natl. Acad. Sci. U.S.A.. 91: 12501-12504; PCTVUS95/ 14692).
  • a fluorescent protein is an Aequorea-related fluorescent protein if any contiguous sequence of 150 amino acids of the fluorescent protein has at least 85% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild-type Aequorea green fluorescent protein (SwissProt Accession No. P42212).
  • the fluorescent protein may be related to Renilla or Phialidium wild-type fluorescent proteins using the same standards.
  • Aequorea-related fluorescent proteins include, for example, wild-type (native) Aequorea victoria GFP, whose nucleotide and deduced amino acid sequences are presented in Genbank Accession Nos. L29345, M62654, M62653 and others Aequorea-related engineered versions of Green Fluorescent Protein, of which some are listed above.
  • P4, P4-3, W7 and W2 fluoresce at a distinctly shorter wavelength than wild type.
  • Recombinant nucleic acid molecules encoding single- or tandem fluorescent protein/polypeptide comprising engineered binding domain, sequences or polypeptides or their binding partners useful in the invention may be expressed either for in vivo assay of the activity of a modifying enzyme on the encoded products.
  • the encoded fusion protiens may be isolated prior to assay, and instead assayed in a cell-free in vitro assay system, as described elsewhere herein.
  • kinases, phosphatases and other modifying enzymes are available commercially (e.g. from Sigma, St. Louis, MO; Promega, Madison, WI; Boehringer Mannheim Biochemicals, Indianapolis, IN; New England Biolabs, Beverly, MA; and others). Alternatively, such enzymes may be prepared in the laboratory by methods well known in the art.
  • the catalytic sub-unit of protein kinase A (c-PKA) can be purified from natural sources (e.g.
  • bovine heart or from cells/organisms engineered to heterologously express the enzyme. Other isoforms of this enzyme may be obtained by these procedures. Purification is performed as previously described from bovine heart (Peters et al.,1977, Biochemistry, 16: 5691-5697) or from a heterologous source (Tsien et al., WO92/00388), and is in each case briefly summarized as follows:
  • Bovine ventricular cardiac muscle (2kg) is homogenized and then centrifuged. The supernatant is applied to a strong anion exchange resin (e.g. Q resin, Bio-Rad) equilibrated in a buffer containing 50mM Tris-HCl, lOmM NaCl, 4mM EDTA pH 7.6 and 0.2mM 2- mercaptoethanol. The protein is eluted from the resin in a second buffer containing 50mM Tris-HCl, 4mM EDTA pH 7.6, 0.2mM 2-mercaptoethanol, 0.5M NaCl. Fractions containing c-PKA are pooled and ammonium sulphate added to 30% saturation.
  • a strong anion exchange resin e.g. Q resin, Bio-Rad
  • Proteins precipitated by this are removed by centrifiigation and the ammonium sulphate concentration of the supernatant was increased to 75% saturation. Insoluble proteins are collected by centrifugation (included c-PKA) and are dissolved in 30mM phosphate buffer pH 7.0, lmM EDTA, 0.2mM 2-mercaptoethanol. These proteins are then dialysed against the same buffer (500 volume excess) at 4°C for two periods of 8 hours each.
  • CM-Sepharose Pharmacia, ⁇ 80 ml resin each
  • Cyclic AMP 10 ⁇ M is added to the material which fails to bind to the CM-Sepharose, and the sample-cAMP mix is incubated with a fresh resin of CM-Sepharose (-100 ml) equilibrated as before.
  • c-PKA is eluted from this column following extensive washing in equilibration buffer by addition of 30mM phosphate pH 6.1 , 1 mM EDTA, 1M KC1, 0.2 mM 2-mercaptoethanol. Fractions containing c-PKA are pooled and concentrated by filtration through a PM-30 membrane (or similar). The c-PKA sample is then subjected to gel-filtration chromatography on a resin such as Sephacryl 200HR (Pharmacia). The purification of recombinant c-PKA is as described in WO 92/00388. General methods of preparing pure and partially-purified recombinant proteins, as well as crude cellular extracts comprising such proteins, are well known in the art.
  • assays of the activity of protein-modifying enzymes may be performed using crude cellular extracts, whether to test the activity of a recombinant protein or one which is found in nature, such as in a biological sample obtained from a test cell line or animal or from a clinical patient.
  • a crude cell extract enables rapid screening of many samples, which potentially finds special application in high- throughput screening methods, e.g. of candidate modulators of protein-modifying enzyme activity.
  • a crude extract with the labeled reporter polypeptide comprising an engineered binding domain, sequence or polypeptide of the invention and a binding partner therefor facilitates easy and rapid assessment of the activity of an enzyme of interest in a diagnostic procedure, e.g., one which is directed at determining whether a protein-modifying enzyme is active at an a physiologically-appropriate level, or in a procedure designed to assess the efficacy of a therapy aimed at modulating the activity of a particular enzyme.
  • Engineered polypeptides, polypeptides comprising an engineered binding domain or sequence or binding partners for such engineered species may be synthesized by Fmoc or Tboc chemistry according to methods known in the art (e.g., see Atherton et al., 1981, J. Chem. Soc. Perkin I, 1981(2): 538-546; Merrifield, 1963, J. Am. Chem. Soc, 85: 2149- 2154, respectively). Following deprotection and cleavage from the resin, peptides are desalted by gel filtration chromatography and analyzed by mass spectroscopy, HPLC, Edman degradation and/or other methods as are known in the art for protein sequencing using standard methodologies.
  • nucleic acid sequences encoding such peptides may be expressed either in cells or in an in vitro transcription/translation system (see below) and, as with enzymes to be assayed according to the invention, the proteins purified by methods well known in the art.
  • phage display in which an engineered binding domain is expressed from a phage chromosome along with on of a library of candidate binding partners. If a candidate binding partner binds the engineered binding domain, both are incorporated into the phage capsid.
  • Engineered binding polypeptides, polypeptides comprising engineered binding domains or sequences, or binding partners therefor are labeled with thiol reactive derivatives of fluorescein and tetramethylrhodamine (isothiocyanate or iodoacetamide derivatives, Molecular Probes, Eugene, OR, USA) using procedures described by Hermanson G.T., 1995, Bioconjugate Techniques, Academic Press, London.
  • primary-amine-directed conjugation reactions can be used to label lysine sidechains or the free peptide N-terminus (Hermason, 1995, supra).
  • Fluorescent peptides are separated from unreacted fluorophores by gel filtration chromatography or reverse phase HPLC.
  • Phosphorylation of engineered binding domains and binding partners in vitro Peptides (0.01-l.O ⁇ M) are phosphorylated by purified c-PKA in 50mM Histidine buffer pH 7.0, 5mM MgSO 4 , lmM EGTA, 0.1-1.0 ⁇ M c-PKA, and 0.2mM [ 3 P] ⁇ -ATP (specific activity ⁇ 2Bq/pmol) at 30-37°C for periods of time ranging from 0 to 60 minutes.
  • the chemistry of the peptide is appropriate (i.e. having a basic charge) the phosphopeptide is captured on a cation exchange filter paper (e.g.
  • Fluorescence measurements of protein modification in vitro in real time Donor and acceptor fluorophore-labeled engineered binding polypeptides or polypeptides comprising engineered binding domains or sequences and the corresponding binding partners for any such engineered molecules (molar equivalents of fluorophore-labeled polypeptide or molar excess of acceptor-labeled polypeptide) are first mixed (if the two are present on separate polypeptides). Samples are analyzed in a fluorimeter using excitation wavelengths relevant to the donor fluorescent label and emission wavelengths relevant to both the donor and acceptor labels. A ratio of emission from the acceptor over that from the donor following excitation at a single wavelength is used to determine the efficiency of fluorescence energy transfer between fluorophores, and hence their spatial proximity.
  • measurements are performed at 0-37 °C as a function of time following the addition of the modifying enzyme (and, optionally, a modulator or candidate modulator of function for that enzyme, as described below) to the system in 50mM histidine pH 7.0, 120 mM KC1, 5mM MgSO 4 , 5mM NaF, 0.05mM EGTA and 0.2mM ATP.
  • the assay may be performed at a higher temperature if that temperature is compatible with the enzyme(s) under study.
  • a cell-free assay system must permit dimerization of an engineered binding domain, sequence or polypeptide with its binding partner to occur in a modification-dependent manner.
  • a system may comprise a low-ionic-strengfh buffer (e.g., physiological salt, such as simple saline or phosphate- and/or Tris-buffered saline or other as described above), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate.
  • physiological salt such as simple saline or phosphate- and/or Tris-buffered saline or other as described above
  • a cell culture medium of which many are known in the art, or a whole or fractionated cell lysate.
  • the components of an assay of post-translational modification of a polypeptide molecule according to the invention may be added into a buffer, medium or lysate or may have been expressed in cells from which a lysate is derived.
  • a cell-free transcription- and/or translation system may be used to deliver one or more of these components to the assay system.
  • Nucleic acids of use in cell-free expression systems according to the invention are as described for in vivo assays, below.
  • An assay of the invention may be peformed in a standard in vitro transcription/translation system under conditions which permit expression of a recombinant or other gene.
  • the TNT ® T7 Quick Coupled Transcription Translation System (Cat.
  • TNT ® Coupled Reticulocyte Lysate Systems (comprising a rabbit reticulocyte lysate) include: TNT ® T3 Coupled Reticulocyte Lysate System (Cat. # L4950; Promega); TNT ® T7 Coupled Reticulocyte Lysate System (Cat.
  • TNT ® SP6 Coupled Reticulocyte Lysate System Cat. # L4600; Promega
  • TNT ® T7/SP6 Coupled Reticulocyte Lysate System Cat. # L5020; Promega
  • TNT ® T7/T3 Coupled Reticulocyte Lysate System Cat. # L5010; Promega.
  • An assay involving a cell lysate or a whole cell may be performed in a cell lysate or whole cell preferably eukaryotic in nature (such as yeast, fungi, insect, e.g., Drosophila), mouse, or human).
  • An assay in which a cell lysate is used is performed in a standard in vitro system under conditions which permit gene expression.
  • a rabbit reticulocyte lysate alone is also available from Promega, either nuclease-treated (Cat. # L4960) or untreated (Cat. # L4151).
  • the invention encompasses methods by which to screen compositions which may enhance, inhibit or not affect (e.g., in a cross-screening procedure in which the goal is to determine whether an agent intended for one purpose additionally affects general cellular functions, of which protein modification is an example) the activity of a protein-modifying enzyme.
  • Candidate modulator compounds from large libraries of synthetic or natural compounds can be screened. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI). Combinatorial libraries are -available and can be prepared.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily produceable by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
  • Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, preferably less than about 750, more preferably less than about 350 daltons. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents.
  • peptide agents may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.
  • an unnatural amino acid such as a D-amino acid, particularly D-alanine
  • Candidate modulators which may be screened according to the methods of the invention include receptors, enzymes, ligands, regulatory factors, and structural proteins.
  • Candidate modulators also include nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens.
  • Candidate modulators additionally comprise proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such as ribozymes or antisense nucleic acids).
  • Proteins or polypeptides which can be screened using the methods of the present invention include hormones, growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structural proteins, viral antigens, parasitic antigens, bacterial antigens and antibodies (see below).
  • Candidate modulators which may be screened according to the invention also include substances for which a test cell or organism might be deficient or that might be clinically effective in higher-than-normal concentration as well as those that are designed to eliminate the translation of unwanted proteins.
  • Nucleic acids of use according to the invention not only may encode the candidate modulators described above, but may eliminate or encode products which eliminate deleterious proteins.
  • Such nucleic acid sequences are antisense RNA and ribozymes, as well as DNA expression constructs that encode them. Note that antisense RNA molecules, ribozymes or genes encoding them may be administered to a test cell or organism by a method of nucleic acid delivery that is known in the art, as described below.
  • Inactivating nucleic acid sequences may encode a ribozyme or antisense RNA specific for the a target mRNA.
  • Ribozymes of the hammerhead class are the smallest known, and lend themselves both to in vitro production and delivery to cells (summarized by Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S; Usman et al.. 1996. Curr. Qpin. Struct. Biol, 6: 527-533).
  • antibodies are of use in the invention as modulators (specifically, as inhibitors) of protein-modifying enzymes.
  • Methods for the preparation of antibodies are well known in the art, and are briefly summarized as follows: Either recombinant proteins or those derived from natural sources can be used to generate antibodies using standard techniques, well known to those in the field. For example, the proteins are administered to challenge a mammal such as a monkey, goat, rabbit or mouse.
  • the resulting antibodies can be collected as polyclonal sera, or antibody-producing cells from the challenged animal can be immortalized (e.g. by fusion with an immortalizing fusion partner) to produce monoclonal antibodies.
  • Monoclonal antibodies e.g. by fusion with an immortalizing fusion partner.
  • the antigen protein may be conjugated to a conventional carrier in order to increases its immunogenicity, and an antiserum to the peptide-carrier conjugate is raised. Coupling of a peptide to a carrier protein and immunizations may be performed as described (Dymecki et al.. 1992, J. Biol. Chem.. 267: 4815-4823).
  • the serum is titered against protein antigen by ELISA (below) or alternatively by dot or spot blotting (Boersma and Van Leeuwen, 1994, J. Neurosci. Methods. 51 : 317).
  • the antiserum may be used in tissue sections prepared as described below. The serum is shown to react strongly with the appropriate peptides by ELISA, for example, following the procedures of Green et al., 1982, Cell, 28: 477-487. 2. Monoclonal antibodies.
  • monoclonal antibodies may be prepared using a candidate antigen whose level is to be measured or which is to be either inactivated or affinity-purified, preferably bound to a carrier, as described by Arnheiter et al., Nature, 294, 278-280 (1981).
  • Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue is introduced. Nevertheless, monoclonal antibodies may be described as being “raised to” or “induced by” a protein. Monoclonal antibody-producing hybridomas (or polyclonal sera) can be screened for antibody binding to the target protein.
  • antibody we include constructions using the binding (variable) region of such an antibody, and other antibody modifications.
  • an antibody useful in the invention may comprise a whole antibody, an antibody fragment, a polyfunctional antibody aggregate, or in general a substance comprising one or more specific binding sites from an antibody.
  • the antibody fragment may be a fragment such as an Fv, Fab or F(ab') 2 fragment or a derivative thereof, such as a single chain Fv fragment.
  • the antibody or antibody fragment may be non-recombinant, recombinant or humanized.
  • the antibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth.
  • an aggregate, polymer, derivative and conjugate of an immunoglobulin or a fragment thereof can be used where appropriate.
  • a candidate modulator of the activity of a protein-modifying enzyme may be assayed according to the invention as described herein, is determined to be effective if its use results in a difference of about 10%> or greater relative to controls in which it is not present (see below) in FRET resulting from the association of labeled engineered binding domains, sequences or polypeptides and their corresponding binding partner(s) in the presence of a protein-modifying enzyme.
  • IndeXcontr o l is the quantitative result (e.g., amount of- or rate of change in fluorescence at a given frequency, rate of molecular rotation, FRET, rate of change in FRET or other index of modification, including, but not limited to, enzyme inhibition or activation) obtained in assays that lack the candidate modulator (in other words, untreated controls), and Indexs am pi e represents the result of the same measurement in assays containing the candidate modulator.
  • control measurements are made with a differentially-labeled engineered binding domain, sequence or polypeptide and its binding partner only and with these molecules plus a protein-modifying enzyme which recognizes a site present on them.
  • Differentially-labeled engineered binding domains, sequences or polypeptides of the invention and their binding partners are delivered (e.g., by micro injection) to cells, such as smooth muscle cells (DDTl) or ventricular cardiac myocytes as previously described (Riabowol et al., 1988, Cold Spring Harbor Symposia on Quantitative Biology. 53: 85-90).
  • DDTl smooth muscle cells
  • ventricular cardiac myocytes as previously described (Riabowol et al., 1988, Cold Spring Harbor Symposia on Quantitative Biology. 53: 85-90).
  • the ratio of emission from the labeled molecule(s) is measured as described above via a photomultiplier tube focused on a single cell.
  • a kinase e.g., PKA by the addition of dibutyryl cAMP or ⁇ -adrenergic agonists
  • a suitable antagonist e.g., cAMP antagonist Rp-cAMPS
  • an ADP ribosylating enzyme may be stimulated with cholera toxin (G-protein recognition feature) or with brefeldin A.
  • Heterologous expression of peptides Engineered binding domains, sequences or polypeptides and their binding partners can be produced from the heterologous expression of DNA sequences which encode them or may be chemically synthesized.
  • Biological expression can be in procaryotic or eukaryotic cells using a variety of plasmid vectors capable of instructing heterologous expression. Purification of these products is achieved by destruction of the cells (e.g. French Press) and chromatographic purification of the products. This latter procedure can be simplified by the inclusion of an affinity purification tag at one extreme of the peptide, separated from the peptide by a protease cleavage site if necessary.
  • the assays of the invention are broadly applicable to a host cell susceptible to transfection or transformation including, but not limited to, bacteria (both gram-positive and gram-negative), cultured- or explanted plant (including, but not limited to, tobacco, arabidopsis, carnation, rice and lentil cells or protoplasts), insect (e.g., cultured Drosophila or moth cell lines) or vertebrate cells (e.g., mammalian cells) and yeast.
  • bacteria both gram-positive and gram-negative
  • cultured- or explanted plant including, but not limited to, tobacco, arabidopsis, carnation, rice and lentil cells or protoplasts
  • insect e.g., cultured Drosophila or moth cell lines
  • vertebrate cells e.g., mammalian cells
  • yeast yeast
  • Organisms are currently being developed for the expression of agents including DNA, RNA, proteins, non-proteinaceous compounds, and viruses.
  • Such vector microorganisms include bacteria such as Clostridium (Parker et al., 1947, Proc. Soc. Exp. Biol. Med., 66: 461-465; Fox et al., 1996, Gene Therapy. 3: 173-178; Minton et al., 1995, FEMS Microbiol. Rev.. 17: 357-364), Salmonella (Pawelek et al., 1997, Cancer Res.. 57: 4537-4544; Saltzman et al., 1996, Cancer Biother. Radiopharm..
  • Dictyosteliida such as of the genera Polysphondylium and Dictystelium, e.g. Dictyostelium discoideum - and Myxomycetes - e.g.
  • Plant cells useful in expressing polypeptides of use in assays of the invention include, but are not limited to, tobacco (Nicotiana plumb aginifolia and Nicotiana tabacum), arabidopsis (Arabidopsis thaliana), Aspergillus niger, Brassica napus, Brassica nigra, Datura innoxia, Vicia narbonensis, Viciafaba, pea (Pisum sativum), cauliflower, carnation and lentil (Lens culinaris). Either whole plants, cells or protoplasts may be transfected with a nucleic acid of choice.
  • Methods for plant cell transfection or stable transformation include inoculation with Agrobacterium tumefaciens cells carrying the construct of interest (see. among others, Turpen et al., 1993, J. Virol. Methods, 42: 227-239), administration of liposome-associated nucleic acid molecules (Maccarrone et al, 1992, Biochem. Biophvs. Res. Commun.. 186: 1417-1422) and microparticle injection (Johnston and Tang, 1993, Genet. Eng. (NY), 15: 225-236), among other methods.
  • a generally useful plant transcriptional control element is the cauliflower mosaic virus (CaMV) 35S promoter (see, for example. Saalbach et al., 1994, Mol. Gen.
  • Non-limiting examples of nucleic acid vectors useful in plants include pGSGLUCl (Saalbach et al., 1994, supra), pGA492 (Perez et al., 1989, Plant Mol. Biol. 13: 365-373), pOCA18 (Olszewski et al., 1988, Nucleic Acids Res.. 16: 10765-10782), the Ti plasmid (Roussell et al., 1988, Mol. Gen. Genet. 211 : 202-209) and pKR612Bl (Balazs et al, 1985, Gene, 40: 343-348).
  • Mammalian cells are of use in the invention. Such cells include, but are not limited to, neuronal cells (those of both primary explants and of established cell culture lines) cells of the immune system (such as T-cells, B-cells and macrophages), fibroblasts, hematopoietic cells and dendritic cells.
  • neuronal cells such as T-cells, B-cells and macrophages
  • fibroblasts such as T-cells, B-cells and macrophages
  • hematopoietic cells e.g. hematopoietic stem cells
  • unseparated hematopoietic cells and stem cell populations may be made susceptible to DNA uptake. Transfection of hematopoietic stem cells is described in Mannion-Henderson et al., 1995, Exp.
  • Nucleic acid vectors for the expression of assay components of the invention in cells or multicellular organisms are provided.
  • a nucleic acid of use according to the methods of the invention may be either double- or single stranded and either naked or associated with protein, carbohydrate, proteoglycan and/or lipid or other molecules.
  • Such vectors may contain modified and/or unmodified nucleotides or ribonucleotides.
  • the gene to be transfected may be without its native transcriptional regulatory sequences, the vector must provide such sequences to the gene, so that it can be expressed once inside the target cell.
  • sequences may direct transcription in a tissue-specific manner, thereby limiting expression of the gene to its target cell population, even if it is taken up by other surrounding cells.
  • sequences may be general regulators of transcription, such as those that regulate housekeeping genes, which will allow for expression of the transfected gene in more than one cell type; this assumes that the majority of vector molecules will associate preferentially with the cells of the tissue into which they were injected, and that leakage of the vector into other cell types will not be significantly deleterious to the recipient mammal. It is also possible to design a vector that will express the gene of choice in the target cells at a specific time, by using an inducible promoter, which will not direct transcription unless a specific stimulus, such as heat shock, is applied.
  • a specific stimulus such as heat shock
  • a gene encoding a component of the assay system of the invention or a candidate modulator of protein-modifying enzyme activity may be transfected into a cell or organism using a viral or non- viral DNA or RNA vector, where non- viral vectors include, but are not limited to, plasmids, linear nucleic acid molecules, artificial chromomosomes and episomal vectors. Expression of heterologous genes in mammals has been observed after injection of plasmid DNA into muscle (Wolff J. A. et al, 1990, Science, 247: 1465-1468; Carson D.A. et al., US Patent No.
  • microbial plasmids such as those of bacteria and yeast, are of use in the invention.
  • pBR322 is useful according to the invention, and pUC is preferred.
  • other plasmids which are useful according to the invention are those which require the presence of plasmid encoded proteins for replication, for example, those comprising pT 181, FII, and FI origins of replication.
  • origins of replication which are useful in assays of the invention in E coli and S typhimurium include but are not limited to, pHETK (Garapin et al., 1981, Proc. Natl. Acad. Sci. U.S.A., 78: 815-819), p279 (Talmadge et al., 1980. Proc. Natl. Acad. Sci.
  • Yeast plasmids Three systems are used for recombinant plasmid expression and replication in yeasts:
  • plasmid Integrating.
  • An example of such a plasmid is Yip, which is maintained at one copy per haploid genome, and is inherited in Mendelian fashion.
  • Such a plasmid containing a gene of interest, a bacterial origin of replication and a selectable gene (typically an antibiotic-resistance marker), is produced in bacteria.
  • the purified vector is linearized within the selectable gene and used to transform competent yeast cells. Regardless of the type of plasmid used, yeast cells are typically transformed by chemical methods (e.g. as described by Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • the cells are treated with lithium acetate to achieve transformation efficiencies of approximately 10 4 colony-forming units (transformed cells)/ ⁇ g of DNA.
  • Yeast perform homologous recombination such that the cut, selectable marker recombines with the mutated (usually a point mutation or a small deletion) host gene to restore function.
  • Transformed cells are then isolated on selective media.
  • ARS-CEN Low copy-number ARS-CEN, of which YCp is an example.
  • a plasmid contains the autonomous replicating sequence (ARS1), a sequence of approximately 700 bp which, when carried on a plasmid, permits its replication in yeast, and a centromeric sequence (CEN4), the latter of which allows mitotic stability. These are usually present at 1-2 copies per cell. Removal of the CEN sequence yields a YRp plasmid, which is typically present in
  • the 2 ⁇ sequence which acts as a yeast replicon giving rise to higher plasmid copy number; however, these plasmids are unstable and require selection for maintenance.
  • Copy number is increased by having on the plasmid a selection gene operatively linked to a crippled promoter. This is usually the LEU2 gene with a truncated promoter (LEU2-d), such that low levels of the Leu2p protein are produced; therefore, selection on a leucine- depleted medium forces an increase in copy number in order to make an amount of Leu2p sufficient for cell growth.
  • LEU2-d truncated promoter
  • yeast plasmids useful in the invention include the YRp plasmids (based on autonomously-replicating sequences, or ARS) and the YEp plasmids (based on the 2 ⁇ circle), of which examples are YEp24 and the YEplac series of plasmids (Gietz and Sugino, 1988, Gene. 74: 527-534).
  • ARS autonomously-replicating sequences
  • YEp plasmids based on the 2 ⁇ circle
  • yeast plasmid sequences typically comprise an antibiotic resistance gene, a bacterial origin of replication (for propagation in bacterial cells) and a yeast nutritional gene for maintenance in yeast cells.
  • the nutritional gene (or "auxotrophic marker") is most often one of the following (with the gene product listed in parentheses and the sizes quoted encompassing the coding sequence, together with the promoter and terminator elements required for correct expression):
  • TRP1 PhosphoADP-ribosylanthranilate isomerase, which is a component of the tryptophan biosynthetic pathway.
  • URA3 Optidine-5'-phosphate decarboxylase, which takes part in the uracil biosynthetic pathway.
  • LEU2 (3-Isopropylmalate dehydrogenase, which is involved with the leucine biosynthetic pathway).
  • HIS3 Imidazoleglycerolphosphate dehydratase, or IGP dehydratase.
  • LYS2 ⁇ -aminoadipate-semialdehyde dehydrogenase, part of the lysine biosynthetic pathway.
  • the screening system may operate in an intact, living multicellular organism, such as an insect or a mammal.
  • Methods of generating transgenic Drosophila mice and other organisms, both transiently and stably, are well known in the art; detection of fluorescence resulting from the expression of Green Fluorescent Protein in live Drosophila is well known in the art.
  • One or more gene expression constructs encoding one or more of a labeled engineered binding domain, sequence or polypeptide, a binding partner therefor, a protein-modifiying enzyme and, optionally, a candidate modulator thereof are introduced into the test organism by methods well known in the art (see also below). Sufficient time is allowed to pass after administration of the nucleic acid molecule to allow for gene expression, for binding of engineered binding domains, sequences or polypeptides and their binding partners, and for chromophore maturation, if necessary (e.g., Green Fluorescent Protein matures over a period of approximately 2 hours prior to fluorescence) before FRET is measured.
  • a reaction component (particularly a candidate modulator of enzyme function) which is not administered as a nucleic acid molecule may be delivered by a method selected from those described below.
  • the amount of each labeled engineered binding domain or binding partner therefor must fall within the detection limits of the fluorescence-measuring device employed.
  • the amount of an enzmye or candidate modulator thereof will typically be in the range of about l ⁇ g - 100 mg kg body weight.
  • the candidate modulator is a peptide or polypeptide, it is typically administered in the range of about 100 - 500 ⁇ g/ml per dose.
  • a candidate modulator is tested in a concentration range that depends upon the molecular weight of the molecule and the type of assay. For example, for inhibition of protein/protein or protein/DNA complex formation or transcription initiation (depending upon the level at which the candidate modulator is thought or intended to modulate the activity of a protein modifying enzyme according to the invention), small molecules (as defined above) may be tested in a concentration range of lpg - 100 ⁇ g/ml, preferably at about 100 pg - 10 ng/ml; large molecules, e.g., peptides, may be tested in the range of 10 ng - 100 ⁇ g/ml, preferably 100 ng - 10 ⁇ g/ml.
  • nucleic acid molecules are administered in a manner compatible with the dosage formulation, and in such amount as will be effective.
  • such an amount should be sufficient to result in production of a detectable amount of the labeled protein or peptide, as discussed above.
  • the amount produced by expression of a nucleic acid molecule should be sufficient to ensure that at least 10% of engineered binding domains or binding partners therefor will undergo modification if they comprise a target site recognized by the enzyme being assayed.
  • the amount of a nucleic acid encoding a candidate modulator of a protein modifying enzyme of the invention must be sufficient to ensure production of an amount of the candidate modulator which can, if effective, produce a change of at least 10%> in the effect of the target protein modifying enzyme on FRET resulting from binding of a engineered binding domain to its binding partner or, if administered to a patient, an amount which is prophylactically and/or therapeutically effective.
  • the dosage to be administered is directly proportional to the amount needed per cell and the number of cells to be transfected, with a correction factor for the efficiency of uptake of the molecules.
  • the strength of the associated transcriptional regulatory sequences also must be considered in calculating the number of nucleic acid molecules er target cell that will result in adequate levels of the encoded product. Suitable dosage ranges are on the order of, where a gene expression construct is administered, 0.5- to l ⁇ g, or 1- lO ⁇ g, or optionally 10- 100 ⁇ g of nucleic acid in a single dose.
  • dosages of up to lmg may be advantageously used.
  • the number of molar equivalents per cell vary with the size of the construct, and that absolute amounts of DNA used should be adjusted accordingly to ensure adequate gene copy number when large constructs are injected. If no effect (e.g., of a modifying enzyme or an inhibitor thereof) is seen within four orders of magnitude in either direction of the starting dosage, it is likely that an enzyme does not recognize the target site of the engineered binding domain (and, optionally, its binding partner) according to the invention, or that the candidate modulator thereof is not of use according to the invention.
  • the concentration must be kept below harmful levels, which may be known if an enzyme or candidate modulator is a drug that is approved for clinical use.
  • Such a dosage should be one (or, preferably, two or more) orders of magnitude below the LD 50 value that is known for a laboratory mammal, and preferably below concentrations that are documented as producing serious, if non-lethal, side effects.
  • Components of screening assays of the invention may be formulated in a physiologically acceptable diluent such as water, phosphate buffered saline, or saline, and further may include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art.
  • Administration of labeled polypeptides comprising a engineered binding domain, sequence, polypeptide or a binding partner therefor, a protein kinase or phosphatase or a candidate modulator as described herein may be either localized or systemic.
  • Localized administration of a component of an assay of the invention is preferably by via injection or by means of a drip device, drug pump or drug- saturated solid matrix from which the nucleic acid can diffuse implanted at the target site.
  • a tissue that is the target of delivery according to the invention is on a surface of an organism, topical administration of a pharmaceutical composition is possible.
  • compositions comprising a composition of- or of use in the invention which are suitable for topical administration can take one of several physical forms, as summarized below: (i) A liquid, such as a tincture or lotion, which may be applied by pouring, dropping or “painting” (i. e. spreading manually or with a brush or other applicator such as a spatula) or injection.
  • a liquid such as a tincture or lotion
  • An ointment or cream which may be spread either manually or with a brush or other applicator (e.g. a spatula), or may be extruded through a nozzle or other small opening from a container such as a collapsible tube.
  • a brush or other applicator e.g. a spatula
  • a dry powder which may be shaken or sifted onto the target tissue or, alternatively, applied as a nebulized spray.
  • a liquid-based aerosol which may be dispensed from a container selected from the group that comprises pressure-driven spray bottles (such as are activated by squeezing), natural atomizers (or "pump-spray” bottles that work without a compressed propellant) or pressurized canisters.
  • a carbowax or glycerin preparation such as a suppository, which may be used for rectal or vaginal administration of a therapeutic composition.
  • the tissue to which a candidate modulator of a protein kinase or phosphatase is to be delivered for assay (or, if found effective, for therapeutic use) is the lung.
  • the route of administration is via inhalation, either of a liquid aerosol or of a nebulized powder of.
  • Drug delivery by inhalation is well known in the art for the treatment of asthma, bronchitis and anaphylaxis.
  • Systemic administration of a protein, nucleic acid or other agent according to the invention may be performed by methods of whole-body drug delivery are well known in the art. These include, but are not limited to, intravenous drip or injection, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug-impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device, which may comprise a reservoir of exogenously-produced protein, nucleic acid or other material or may, instead, comprise cells that produce and secrete a engineered binding domain and/or a binding partner therefor, modifying enzyme or candidate modulator thereof.
  • injection may be performed either by conventional means (i.e. using a hypodermic needle) or by hypospray (see Clarke and Woodland, 1975, Rheumatol. RehabiL. 14: 47-49).
  • Components of assays of the invention can be given in a single- or multiple dose.
  • Delivery of a nucleic acid may be performed using a delivery technique selected from the group that includes, but is not limited to, the use of viral vectors and non-viral vectors, such as episomal vectors, artificial chromosomes, liposomes, cationic peptides, tissue-specific cell transfection and transplantation, administration of genes in general vectors with tissue- specific promoters, etc.
  • a delivery technique selected from the group that includes, but is not limited to, the use of viral vectors and non-viral vectors, such as episomal vectors, artificial chromosomes, liposomes, cationic peptides, tissue-specific cell transfection and transplantation, administration of genes in general vectors with tissue- specific promoters, etc.
  • kits according to the invention i. A kit for assaying the activity of a protein-modifying enzyme
  • kits which contain the essential components for screening the activity of a an enzyme which mediates a change in protein modification, as described above.
  • a labeled, engineered binding domain, sequence or polypeptide, as defined above, and a differentially labeled binding partner which binds it specifically in a modification-dependent manner is provided, as is a suitable reaction buffer for in vitro assay or, alternatively, cells or a cell lysate.
  • a reaction buffer which is "suitable” is one which is permissive of the activity of the enzyme to be assayed and which permits modification dependent binding of the engineered binding domain, sequence or polypeptide and the binding partner.
  • the labeled components are provided as peptide/protein or a nucleic acid comprising a gene expression construct encoding the one or more of a peptide/protein, as discussed above.
  • Polypeptides in a kit of the invention are supplied either in solution (preferably refrigerated or frozen) in a buffer which inhibits degradation and maintains biological activity, or are provided in dried form, i.e., lyophilized. In the latter case, the components are resuspended prior to use in the reaction buffer or other biocompatible solution (e.g.
  • the resuspension buffer should not inhibit modification-dependent protein binding when added to the reaction buffer in an amount necessary to deliver sufficient protein for an assay reaction.
  • Polypeptides provided as nucleic acids are supplied- or resuspended in a buffer which permits either transfection/transformation into a cell or organism or in vitro transcription/translation, as described above.
  • kits include cells.
  • Eukaryotic or prokaryotic cells as described above, are supplied in- or on a liquid or solid physiological buffer or culture medium (e.g. in suspension, in a stab culture or on a culture plate, e.g. a Petri dish).
  • the cells are typically refrigerated, frozen or lyophilized in a bottle, tube or vial.
  • An enzyme being assayed according to the invention is added to the assay system either as a protein (isolated, partially-purified or present in a crude preparation such as a cell extract or even a living cell) or a recombinant nucleic acid.
  • Methods of expressing a nucleic acid comprising an enzyme or other protein are well known in the art (see again above).
  • An assay of the invention is carried out using the kit according to the methods described above, in the Example and elsewhere.
  • kits for screening a candidate modulator of protein-modifying enzyme activity A candidate modulator of post-translational modification may be assayed using a kit of the invention.
  • a kit as described above is used for this application, with the assay performed further comprising the addition of a candidate modulator of the modifying enzyme which is present to the reaction system.
  • a protein-modifying enzyme is supplied with the kit, either as a protein or nucleic acid as described above.
  • Assays of protein activity are performed as described above. At a minimum, three detections are performed, one in which the engineered binding domain, sequence or polypeptide and its binding partner are present without the modifying enzyme or candidate modulator thereof (control reaction A), one in which the same polypeptide components are incubated with the modifying enzyme under conditions which permit the modification reaction to occur (control reaction B) and one in which the modifying enzyme and candidate inhibitor are both incubated with the labeled engineered binding domain, sequence or polypeptide and corresponding binding partner under conditions which permit the modification reaction to occur (test reaction).
  • control reaction A one in which the engineered binding domain, sequence or polypeptide and its binding partner are present without the modifying enzyme or candidate modulator thereof
  • control reaction B one in which the same polypeptide components are incubated with the modifying enzyme under conditions which permit the modification reaction to occur
  • test reaction one in which the modifying enzyme and candidate inhibitor are both incubated with the labeled engineered binding domain, sequence or polypeptide and corresponding binding partner under conditions which permit the modification reaction to occur
  • Example 1 Generation of an engineered binding domain/binding partner pair for use in a protein binding assay of enzymatic activity (cAMP dependent protein kinase) according to the invention.
  • a test protein may be expressed as an extension of the g3p protein, while a target protein (polypeptide A) for the test protein is fused to a convenient affinity tag (such as His6) and expressed in the same phage.
  • a convenient affinity tag such as His6
  • the binding of polypeptide A to the test protein displayed on g3p facilitates the affinity purification of the phage particle.
  • the binding partner structures can be derived from heterodimeric protein complexes, homodimeric protein complexes or from two domains of a single polypeptide which have been cleaved into separate entities for the purpose of this invention.
  • ubiquitin a 76-residue cellular protein of known tertiary structure
  • Ub ubiquitin
  • the protein backbone of ubiquitin (Ub) can be broken at one of a number of locations without significant loss of structure. In this cleaved state, the two halves of the ubiquitin protein remain together in a compact and stable complex (Johnsson and Varshavsky, 1994, Proc. Natl. Acad. Sci U.S.A., 91 : 10340-10344).
  • sUb-N for the N-terminal fragment of the split ubiquitin
  • sUb-C for the C-terminal fragment of split ubiquitin
  • Mammalian Ub has been cloned into a phage-display vector system, pCANTABb, developed at the University of London from the Pcanbab5 VECTOR. Ub can be expressed from this system and displayed on the surface of filamentous phage (Finucane et al (1999) Biochemistry 36 1 1604-11612 Finucane & Woolfson (1999) Biochem 36 11613-11623).
  • This vector system has been used to prepare constructs that direct the expression of the sUb fragments and, in turn, these are used to generate the required mutants.
  • the split has been positioned at Asp39, which is a highly exposed residue in the native Ub structure.
  • Residues 20-23 of sUb-N, which have the sequence SDTI are be mutated to RRKS to be recognised and phosphorylated to PKA.
  • the SD to RR mutations should not destabilize the sUb complex significantly, because in the native structure, these residues are highly exposed to solvent.
  • the 123 to S mutation is expected to be destabilizing, because 123 is a hydrophobic residue located in the core of the native protein. Thus, in addition to the chain break, this mutation may destabilize the sUb sufficiently to prevent complexation of sUb-N and sUb-C.
  • residues Leu43, Leu50 and Leu56 of sUb-C which are also in the core of the protein and contact 123 in native ubiquitin, are mutated to all combinations of the 20 amino acids to give a library of 8,000 mutations.
  • Stable sUb complexes are selected from this pool as follows: sUb- ⁇ containing the RRKS mutation are expressed with an ⁇ -terminal hexahistidine tag to allow it to be captured onto surfaces with chelated ⁇ i, such as a BIAcore chip used in surface plasmon resonance or ⁇ i-agarose beads.
  • the mutant sUb-Cs are made as a fusion with g3p to allow them to be displayed on surface of filamentous phage.
  • the separated His6-sUb- ⁇ :sUb-C-phage complexes could then be broken using protein denaturants such as urea or guanidinium chloride that do not disrupt the phage. This are permit elution of the phage, which are then be amplified by re-infection into E-coli. This panning and amplification procedure is repeated a number of times in order to amplify mutant sUb-Cs that bind tightly to the sUb-N mutant and rescue sUb complexation. The selected mutants are identified by sequencing of the selected phage DNA.
  • S'-AATCTCGAGACCGCCGCCCCTCAGGCGGAGGAC 3' are used to add 5' (Pstl) and 3' (Xhol) restriction sites and terminal glycine codons.
  • the PCR product was ligated into the pGEM-T vector (Promega) and sub-cloned into an in-house vector, pCANTABb, using the Pstl and Xhol sites.
  • pCANTABb was derived from the phage-display vector pCANTAB5 by adding the coding sequence for a hexahistidine tag 5' to the g3p sequence (McCafferty et al, 1994, Appl. Biochem. Biotech., 47: 157-173).
  • the pCANTABb-Ub construct directed the expression of the protein fusion: g3p periplasmic leader sequence-hexahistidine tag- LQG-Ub section 1 ; and the independent production of gill periplasmic leader sequence-Ub-GLDQQ-g3p.
  • the construct for sUb-N was prepared from pCANTABb-Ub by eliminating the C-terminal half of the protein Asp39-Gly76, using Kunkel mutagenesis (Kunkel, 1985, Proc. Natl. Acad. Sci. U.S.A., 82: 488-492) and the mutagenic primer 5'-TGCGGCCGCCTACTAAGGGGGGATGCCCTC-3'.
  • sUb-C The construct for sUb-C was prepared from pCANTABb-Ub by sticky-feet mutagenesis (Clackson and Winter, 1989, Nucleic Acids Res., 17(24): 10163-10170) using the primer 5-GAGCCTCTGCTGGTCGGCCATGGCCGGCTG-3', which effectively excises the DNA coding sUb-N. Mutagenesis of Ub-N to insert PKA phosphorylation site
  • Kunkel mutagenesis (Kunkel, 1985, supra) is used with the following mutagenic primer:
  • Phage for biopanning and analysis are prepared as follows: XL 1 -blue E-coli cells, transformed with appropriate vectors, are superinfected with Ml 3 helper phage and grown overnight at 37 C. Phage are precipitated from the growth supernatant using 20%>PEG/2.5M NaCl, resuspended in 1000 ml of buffer A (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and clarified briefly by centrifugation. For the selection studies on the phage library, four rounds of panning are typically performed. In general, conditions for panning are established using surface plasmon resonance in BIAcore.
  • Ni-derivatized NTA chips are used for this purpose along with standard running buffers and protocols. After the chips are derivatized with Ni, purified His6-sUb-N carrying the PKA recognition site is added and bound to the chip surface. Phage displaying the library of sUb-C mutants are passed over the fully derivatized chip and binding (sUb complex formation) is monitored by changes in the SPR signal. This method is also useful in preparative phage selection; however, for displayed libraries with more mutants, preparative panning is done on Ni-agarose beads. A typical protocol for this is: 100 ml of phage-library preparations are mixed with 800 ml of buffer and 250 ml of Ni-NTA agarose beads.
  • Phage are allowed to bind to the beads for 10 minutes at room temperature. Beads are then washed a number of times (e.g. at least 5) with 750 ml of buffer, and the supernatant removed by centrifiigation at each stage. Phage bound to the beads are then eluted with buffer containing 250 mM imidazole, and amplified and purified as described above. The sequences of the selected sUb-C sequences that bind the mutant sUb-N peptide are determined from amplified phage by isolating and sequencing the phagemid DNA.
  • binding partners identified by phage display in an assay of the invention
  • the component polypeptides of a 'binding pair' comprising an engineered binding domain and its binding partner, such as those identified through phage display as above, are expressed and purified by molecular and biochemical known to one of ordinary skill in the art.
  • At least one of the engineered binding domain and the binding partner is labelled with a detectable label, as described above.
  • the engineered binding domain is contacted with the binding partner in a buffer or other medium which permits modification-dependent proteimprotein binding (binding that occurs specifically when the site for post-translational modification is in one modification state but not the other).
  • Methods by which to assess proteimprotein binding are performed, both in the presence and absence of modifying enzyme, candidate modifying enzyme (i.e., an enzyme of unknown function) or a biological sample whose enzymatic activity is assayed according to the methods of the invention.
  • modifying enzyme i.e., an enzyme of unknown function
  • candidate modifying enzyme i.e., an enzyme of unknown function
  • a biological sample whose enzymatic activity is assayed according to the methods of the invention.
  • the engineered binding domain which is not labelled with a detectable label, is immobilized and then contacted with the binding partner, where the partner is still attached to a phage particle from the phage display procedure.
  • Example 2 - PKA assay using engineered ZAP70 SH2 domain and binding partner derived from T-cell receptor zeta chain The assay described herein is based on the concept that the tandem SH2 domain from
  • ZAP binds the dually tyrosine phosphorylated motif from the zeta chain of the T-cell receptor (TCR ⁇ ). Binding only occurs when the TCR ⁇ is phosphorylated on both tyrosine residues, i.e. the interaction between one phospho-tyrosine and one of the tandem SH2 domains is not enough to give stringent binding. Immobilised assays have been performed to demonstrate binding of a TCR peptide to the tandem SH2 domain of ZAP.
  • a protein kinase A (PKA) site is introduced into the ZAP protein, in close proximity to one of the phospho-tyrosine binding pockets, to enable phosphorylation by PKA.
  • PKA protein kinase A
  • the resultant PCR fragment is digested with Ndel and EcoRl and inserted into pET28a (Novagen) to generate vector pFS45.
  • DNA encoding GFP in the vector pQBI25-FNI (Quantum) is digested with M and the resultant, single stranded 5' overhang is "filled in” using T4 DNA polymerase (NEB) to generated complete, double stranded DNA.
  • NEB T4 DNA polymerase
  • the vector pFS45 is digested with Hindlll and the resultant 5' overhang is "filled in” with T4 DNA polymerase and then further digested with EcoRl. After the digested vector is gel purified it is ligated with the purified DNA encoding GFP to generate pFS46, which is designed to express a ZAP70-GFP fusion protein in bacteria.
  • LB/kanamycin (lOO ⁇ g/ml).
  • the starter cultures are incubated overnight at 37°C with shaking. From these starter cultures 1ml is used to inoculate 400ml Terrific Brofh/kanamycin (lOO ⁇ g/ml) in a 2L, baffled flask. Cultures are incubated at 37°C at 200 rpm for approximately 5 hrs until the OD 600nm had reached 0.5 Abs units. At this point cultures are induced by adding IPTG to a concentration of lmM. The cultures are then left incubating at room temperature overnight with gentle shaking on a benchtop rotator.
  • Bacteria are harvested by centrifugation at 3000 rpm for 20 min.
  • the bacterial pellet is resuspended in 25ml lysis buffer (50mM Pi pH 7.0, 300mM NaCl, 2% Proteinase inhibitor cocktail (Sigma), 0.75mg ml Lysozyme). Lysis of the resuspended cells is initiated by gentle stirring for 1 hr. at room temperature.
  • the partially lysed mixture is subjected to 2 cycles of freeze thawing in liquid nitrogen.
  • the cells are sonicated on ice using a 10mm probe at high power. Sonication is performed on a pulse setting for a period of 3 min.
  • the crude lysate is then centrifuged at 15000 rpm for 30 min.
  • His tagged proteins are purified from the clear lysate using TALON® resin (Clontech). Proteins are bound to the resin in a batchwise manner by gentle shaking at room temperature for 30 min. Non-His tagged proteins are removed by washing the resin at least twice with lOx bed volume of wash buffer (50mM sodium phosphate pH 7.0, 300mM NaCl, 5mM fluorescence-blank Imidazole). The washed resin was loaded into a 2 ml column and the bound proteins released with elution buffer (50mM sodium phosphate pH 7.0, 300mM NaCl, 150mM florescence-blank Imidazole). Elution is normally achieved after the first 0.5ml and within 2-3ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10% glycerol.
  • Biotinylated peptides are immobilised in a 96-well streptavidin coated plate
  • peptide (200ml) is incubated in the wells for lhr at room temperature with gentle agitation on a rotating platform. Excess peptide is removed with 3 washes of 200 ml of TBST.
  • ZAPPKA-GFP Vector construction To introduce a PKA site within ZAP in close proximity to one of the tyrosine binding pockets, residues 207-210 (TVYH) are mutated to RRAS. The following oligos are used in the mutagenesis:
  • Reverse primer 2 GGGGCTAGCGCGCCGCTTCCC ATAGATGAGGG These oligos used in tandem with the original cloning oligos create two ZAP fragments which can be joined together by the Nhel site (indicated in bold) present in the primer 2 set. The ZAPPKA fragment is then inserted into pFS46 to generate a vector which is designed to express a ZAPPKA-GFP fusion protein in bacteria.
  • Proteins are purified as described above for ZAP-GFP.
  • Immobilized assay The immobilized assay is performed as described above. It is first necessary to demonstrate that ZAPPKA-GFP binds in an equivalent manner to ZAP-GFP. Once this is confirmed, ZAP-GFP and ZAPPKA-GFP are pre-phosphorylated using 1.5pmol of PKA in 200 ml PKA buffer (50mM Histidine buffer pH 7.0, 5mM MgSO4, 120mM KC1, 5mM NaF, lmM ATP, 0.2mg/ml BSA) for 1 hr at 30°C. After phosphorylation, the immobilized assays are performed as before.
  • PKA buffer 50mM Histidine buffer pH 7.0, 5mM MgSO4, 120mM KC1, 5mM NaF, lmM ATP, 0.2mg/ml BSA
  • Pre-phosphorylation is expected to have no effect on ZAP-GFP binding but is contemplated to completely inhibit binding of ZAPPKA-GFP to the immobilized TCR ⁇ peptide.
  • the presence of the phosphorylated peptide is detected by the fluorescence of GFP as described above.
  • binding will be detected for the ZAP-GFP and the non-phosphorylated ZAPPKA-GFP, while the PKA-phosphorylated ZAPPKA-GFP will display little to no binding.
  • Engineered binding domain assay using an immobilised assay with a natural binding partner labelled with a coiled-coil tag and a fluorescent detector molecule (ZAP70-FJ-fluorescein peptide).
  • the ZAP70SH2 domain is constructed as a fusion protein with a coiled-coil peptide based on the Fos/Jun coiled-coil peptide. Oligonucleotides based on the coiled-coil domain of Fos/Jun have been designed and synthesised: Forward primer:
  • the primers are annealed together by heating to 96°C followed by slow cooling to room temperature.
  • Complete double stranded DNA is generated by "filling in” the single stranded 5' overhangs using Klenow fragment of DNA polymerase I (NEB).
  • the DNA fragment is purified by electrophoresis in 1.2% agarose gel and DNA is extracted from an isolated gel band using Qiagen spin columns.
  • the purified fragment is digested with Sad and Xhol and purified as above prior to ligation into the bacterial expression vector pET28a to generate vector FS101.
  • FS101 is digested with Nhel and EcoRI and DNA encoding ZAPPKA is ligated into this plasmid to generate a vector that is designed to express ZAPPKA-Fos/Jun.
  • the ZAPPKA-FJ purification and labelling with fluorescein is according to the methods described above for ZAP GFP except that the IPTG induction is at room temperature for 90 mins.
  • RMRQLEDRVEELREQNWHLANQVARLRQRVCELKARV Peptide domains can be specifically labeled on amine or thiol groups with chemical fluorophores such as fluorescein or rhodamine. Fluorophores with thiol or amine reactive chemistries are readily available from commercial sources such as Molecular Probes. These fluorophores can be conjugated to peptides under mild conditions (e.g. 20mM TES pH 7 for thiol directed labeling, or 200mM sodium bicarbonate pH 8.3 for amine directed labeling, using 230 ⁇ M peptide in the presence of 200 ⁇ M label).
  • mild conditions e.g. 20mM TES pH 7 for thiol directed labeling, or 200mM sodium bicarbonate pH 8.3 for amine directed labeling, using 230 ⁇ M peptide in the presence of 200 ⁇ M label).
  • the peptide is labeled with fluorescein through amine directed labeling.
  • Purified ZAPPKA-FJ is mixed with the peptide and the mixture morritored by FP to detect coiled-coil formation between the F and J peptides, resulting in ZAPPKA labeled with fluorescein.
  • the immobilized assay is then performed according to the procedure described above.
  • ZAP-GFP ZAP-GFP. Once this is confirmed, ZAP-GFP and ZAPPKA-FJ are pre-phosphorylated using
  • binding will be detected for the ZAP-GFP and the non-phosphorylated ZAPPKA-FJ, while the PKA-phosphorylated ZAPPKA-FJ will display little to no binding.
  • the invention is useful in monitoring the activity of a protein-modifying enzyme. whether the protein is isolated, partially-purified, present in a crude preparation or present in a living cell.
  • the invention is further useful in assaying a cell or cell extract for the presence- or level of activity of a protein modifying enzyme.
  • the invention is additionally useful in assaying the activity of naturally-occurring (mutant) or non-natural (engineered) isoforms of known protein modifying enzymes or, instead, that of novel (natural or non-natural) enzymes.
  • the invention is of use in assaying the efficacy of candidate modulators of the activity of a protein modifying enzyme in inhibiting or enhancing the activity of that enzyme; moreover, is useful to screen potential therapeutic drugs for activity against cloned and/or purified enzymes that may have important clinical pathogenicities when mutated.
  • the invention is further of use in the screening of candidate bioactive agents (e.g., drugs) for side effects, whereby the ability of such an agent to modulate the activity of a protein modifying enzyme may be indicative a propensity toward provoking unintended side-effects to a therapeutic or other regimen in which that agent might be employed.

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Abstract

This invention relates to methods and compositions for monitoring enzymatic activity as a function of the interaction of binding partners, wherein binding is dependent upon addition or subtraction of a chemical moiety to or from one of the binding partners by a protein modifying enzyme.

Description

COMPOSITIONS AND METHODS FOR MONITORING THE MODIFICATION OF ENGINEERED BINDING PARTNERS
FIELD OF THE INVENTION
The invention relates to monitoring of the post-translational modification of a protein.
BACKGROUND OF THE INVENTION
The post-translational modification of proteins have been known for over 40 years and since then has become a ubiquitous feature of protein structure. The addition of biochemical groups to translated polypeptides has wide-ranging effects on protein stability, protein secondary/tertiary structure, enzyme activity and in more general terms on the regulated homeostasis of cells. Such modifications include, but are not limited to, the addition of a phosphate (phosphorylation), carbohydrate (glycosylation), ADP-ribosyl (ADP ribosylation), fatty acid (prenylation, which includes but is not limited to: myristoylation and palmitylation), ubiquitin (ubiquitination) and sentrin (sentrinization; a ubiquitination-like protein modification). Additional examples of post-translational modification include methylation, actylation, hydroxylation, iodination and flavin linkage. Many of the identified modifications have important consequences for the activity of those polypeptides so modified.
Phosphorylation is a well-studied example of a post-translational modification of protein. There are many cases in which polypeptides form higher order tertiary structures with like polypeptides (homo-oligomers) or with unlike polypeptides (hetero-oligomers). In the simplest scenario, two identical polypeptides associate to form an active homodimer. An example of this type of association is the natural association of myosin II molecules in the assembly of myosin into filaments.
The dimerization of myosin II monomers is the initial step in seeding myosin filaments. The initial dimerization is regulated by phosphorylation the effect of which is to induce a conformational change in myosin II secondary structure resulting in the folded 10S monomer subunit extending to a 6S molecule. This active molecule is able to dimerize and subsequently to form filaments. The involvement of phosphorylation of myosin II in this priming event is somewhat controversial. Although in higher eukaryotes the conformational change is dependent on phosphorylation, in Ancanthoamoeba, a lower eukaryote, the post- translational addition of phosphate is not required to effect the initial dimerization step. It is of note that the dimerization domains in myosin II of higher eukaryotes contain the sites for phosphorylation and it is probable that phosphorylation in this region is responsible for enabling myosin II to dimerize and subsequently form filaments. In Dictyostelium this situation is reversed in that the phosphorylation sites are outside the dimerization domain and phosphorylation at these sites is required to effect the disassembly of myosin filaments. In contrast to both these examples, Acanthoamoeba myosin II is phosphorylated in the dimerization domain but this modification is not necessary to enable myosin II monomers to dimerize in this species.
By far the most frequent example of post-translational modification is the addition of phosphate to polypeptides by specific enzymes known as protein kinases. These enzymes have been identified as important regulators of the state of phosphorylation of target proteins and have been implicated as major players in regulating cellular physiology. For example, the cell-division-cycle of the eukaryotic cell is primarily regulated by the state of phosphorylation of specific proteins the functional state of which is determined by whether or not the protein is phosphorylated. This is determined by the relative activity of protein kinases which add phosphate and protein phosphatases which remove the phosphate moiety from these proteins. Clearly, dysfunction of either the kinases or phosphatases may lead to a diseased state. This is best exemplified by the uncontrolled cellular division shown by tumor cells. The regulatory pathway is composed of a large number of genes that interact in vivo to regulate the phosphorylation cascade that ultimately determines if a cell is to divide or arrest cell division.
Currently there are several approaches to analyzing the state of modification of target proteins in vivo:
1. In vivo labelling of cellular substrate pools with radioactive substrate or substrate precursor molecules to result in incorporation of labeled (for example, radiolabeled) moieties (e.g., phosphate, fatty acyl (including, but not limited to, myristoyl, palmityl, sentrin, methyl, actyl, hydroxyl, iodine, flavin, ubiquitin or ADP-ribosyls), which are added to target proteins. Analysis of modified proteins is typically performed by electrophoresis and autoradiography, with specificity enhanced by immunoprecipitation of proteins of interest prior to electrophoresis. 2. Back-labeling. The enzymatic incorporation of a labeled (including, but not limited to, with a radioactive and fluorescent label) moiety into a protein in vitro to estimate the state of modification in vivo.
3. Detection of alteration in electrophoretic mobility of modified protein compared with unmodified (e.g., glycosylated or ubiquitinated) protein.
4. Thin-layer chromatography of radiolabelled fatty acids extracted from the protein of interest.
5. Partitioning of protein into detergent-rich or detergent layer by phase separation, and the effects of enzyme treatment of the protein of interest on the partitioning between aqueous and detergent-rich environments.
6. The use of cell-membrane-permeable protein-modifying enzyme inhibitors (e.g., Wortmannin, staurosporine) to block modification of target proteins and comparable inhibitors of the enzymes involved in other forms of protein modification (above).
7. Antibody recognition of the modified form of the protien (e.g., using an antibody directed at ubiquitin or carbohydrate epitopes), e.g., by Western blotting, of either 1- or 2- dimensional gels bearing test protein samples.
8. Lectin-protein interaction in Western blot format as an assay of the presence of particular carbohydrate groups (defined by the specificity of the lectin in use).
9. The exploitation of eukaryotic microbial systems to identify mutations in protein- modifying enzymes.
These strategies have certain limitations. Monitoring states of modification by pulse or steady-state labelling is merely a descriptive strategy to show which proteins are modified when samples are separated by gel electrophoresis and visualized by autoradiography. This is unsatisfactory, due to the inability to identify many of the proteins that are modified. A degree of specificity is afforded to this technique if it is combined with immunoprecipitation; however, this is of course limited by the availability of antibodies to target proteins. Moreover, only highly-expressed proteins are readily detectable using this technique, which may fail to identify many low-abundance proteins, which are potentially important regulators of cellular functions. The use of enzyme inhibitors to block activity is also problematic. For example, very few kinase inhibitors have adequate specificity to allow for the unequivocal correlation of a given kinase with a specific kinase reaction. Indeed, many inhibitors have a broad inhibitory range. For example, staurosporine is a potent inhibitor of phospholipid/Ca+2 dependant kinases. Wortmannin is some what more specific, being limited to the phosphatidylinositol-3 kinase family. This is clearly unsatisfactory because more than one biochemical pathway may be affected during treatment making the assignment of the effects almost impossible. Finally, yeast (Saccharomyces cervisiae and Schizosaccharomyces pombe) has been exploited as a model organism for the identification of gene function using recessive mutations. It is through research on the effects of these mutations that the functional specificities of many protein-modifying enzymes have been elucidated. However, these molecular genetic techniques are not easily transferable to higher eukaryotes, which are diploid and therefore not as genetically tractable as these lower eukaryotes.
A non-limiting example of post-translational modification is provided by the Ras proteins, which are a conserved group of polypeptides located at the plasma membrane which exist in either a GTP-bound active state or in a GDP-bound inactive state. This family of proteins operates in signal transduction pathways that regulate cell growth and differentiation. In higher eukaryotes, Ras is a key regulator that mediates signal transduction from cell surface tyrosine kinase receptors to the nucleus via activation of the MAP kinase cascade. Recent studies have demonstrated that Ras directly binds a serine/threonine kinase, Raf-1, a product of the c-raf-1 proto-oncogene, and that this association leads to stimulation of the activity of Raf-1 to phosphorylate MAP kinase kinase (MEK). Another post-translational modification is the addition of ubiquitin to selected polypeptides. This provides a key mechanism by which to control the abundance of important regulatory proteins, for example, Gl and mitotic cyclins and the p53 tumor suppressor protein. Ubiquitin is a highly conserved 76-amino-acid cellular polypeptide. The role of ubiquitin in targeting proteins for degradation involves the specific ligation of ubiquitin to the ε group of lysine residues in proteins that are to be degraded or internalized from the plasma membrane. The ubiquitin tag determines the fate of the protein and results in its selective proteolysis. Recently a number of factors have been isolated and shown to be involved in the ubiquitination process.
The initial step in the addition of ubiquitin to a protein is the activation of ubiquitin by the ubiquitin activating enzyme, El . This is an ATP-dependent step resulting in the formation of a thioester bond between the carboxyl terminal glycine of ubiquitin and the active site cysteine residue of El . Activated ubiquitin then interacts with a second factor, the E2 protein. A thioester bond forms between the activated glycine residue of ubiquitin and a cysteine residue in a specific E2 protein. The E2 proteins represent a family of closely- related proteins encoded by different genes that confer specificity in the proteolytic process. The ligation of ubiquitin to target proteins is effected by the involvement of a further factor, a ubiquitin ligase, E3, of which a number are known (in yeast, reviewed by Haas and Siepmann. 1997, FASEB J.. 11 : 1257-1268; in humans, see Honda et al., 1997, FEBS Lett.. 420: 25-27). E3 completes the final step of ubiquitination by attaching ubiquitin via the ε amino group on lysine residues in proteins to be targeted for degradation. Moreover, E3 is able to add ubiquitin to ubiquitin molecules already attached to target proteins, thereby resulting in polyubiquitinated proteins that are ultimately degraded by the multi-subunit proteasome.
An example of heterodimer association is described in patent application number WO92/00388. It describes an adenosine 3: 5 cyclic monophosphate (cAMP) dependent protein kinase which is a four-subunit enzyme being composed of two catalytic polypeptides (C) and two regulatory polypeptides (R). In nature the polypeptides associate in a stoichiometry of R2C2. In the absence of cAMP the R and C subunits associate and the enzyme complex is inactive. In the presence of cAMP the R subunit functions as a ligand for cAMP resulting in dissociation of the complex and the release of active protein kinase. The invention described in WO92/00388 exploits this association by adding fluorochromes to the R and C subunits.
The polypeptides are labeled (or 'tagged') with fluorophores having different excitation/emission wavelengths. The emission from one such fluorophore following excitation effects a second excitation/emission event in the second fluorophore. By monitoring the fluorescence emission or absorption of each fluorophore, which reflects the presence or absence of fluorescence energy transfer between the two, it is possible to derive concentration of cAMP as a function of the association between the R and C subunits. Therefore, the natural affinity of the C subunit for the R subunit has been exploited to monitor the concentration of a specific metabolite, namely cAMP.
The prior art teaches that intact, fluorophore-labeled proteins can function as reporter molecules for monitoring the formation of multi-subunit complexes from protein monomers; however, in each case, the technique relies on the natural ability of the protein monomers to associate. Tsien et al. (WO97/28261) teach that fluorescent proteins having the proper emission and excitation spectra that are brought into physically close proximity with one another can exhibit fluorescence resonance energy transfer ("FRET"). The invention of WO97/28261 takes advantage of that discovery to provide tandem fluorescent protein constructs in which two fluorescent protein labels capable of exhibiting FRET are coupled through a linker to form a tandem construct. In the assays of the Tsien et al. application, protease activity is monitored using FRET to determine the distance between fluorophores controlled by a peptide linker and subsequent hydrolysis thereof. Other applications rely on a change in the intrinsic fluorescence of the protein as in the kinase assays of WO98/06737. The present invention instead encompasses the use of FRET or other detection procedures to monitor the association of polypeptides, as described herein, which are labeled with fluorescent labels (protein and chemical); in the invention, FRET, fluorescence correlation spectroscopy, fluorescence anisotropy, monomeπexcimer fluorescence or other techniques indicate the proximity of two labeled polypeptide binding partners, which labeled partners associate either in the presence or absence of a given post-translational modification to an engineered site which has been introduced into at least one of the partners, but not into the fluorophore, reflecting the modification state of one or both of the binding partners and, consequently, the level of activity of a protein-modifying enzyme. The invention further provides methods which employ non-fluorescent labels including, but not limited to, radioactive labels. In addition, the invention encompasses methods which do not employ detectable labels; such methods include, but are not limited to, the detection of the inhibition or reconstitution of enzymatic activity, which inhibition or reconstitution results from modification-dependent binding or dissociation between an engineered binding domain and a binding partner therefor. There is a need in the art for efficient means of monitoring and/or modulating post- translational protein modification. Further, there is a need to develop a technique whereby the addition/removal of a modifying group can be monitored continuously during real time to provide a dynamic assay system that also has the ability to resolve spatial information. SUMMARY OF THE INVENTION
The invention provides engineered binding domains, sequences and polypeptides, all as defined below, as well as kits comprising these molecules and assays of enzymatic function in which they are employed as reporter molecules.
One aspect of the invention is an isolated engineered binding domain and a binding partner therefor, wherein the isolated engineered binding domain includes a site for post- translational modification and binds the binding partner therefor in a manner dependent upon modification of the site. The invention additionally encompasses a method for monitoring activity of an enzyme comprising performing a detection step to detect binding of an isolated engineered binding domain and a binding partner therefor as a result of contacting one or both of the isolated engineered binding domain and the binding partner with the enzyme, wherein the isolated engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site and wherein detection of binding of the isolated engineered binding domain and the binding partner as a result of the contacting is indicative of enzyme activity.
Another aspect of the invention is a method for monitoring activity of an enzyme comprising performing a detection step to detect dissociation of an isolated engineered binding domain from a binding partner therefor as a result of contacting one or both of the isolated engineered binding domain and the binding partner with the enzyme, wherein the isolated engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site and wherein detection of dissociation of the isolated engineered binding domain from the binding partner as a result of the contacting is indicative of enzyme activity.
As used herein, the term "binding domain" refers in a three-dimensional sense to the amino acid residues of a first polypeptide required for modification-dependent binding between the first polypeptide and its binding partner. The amino acids of a "binding domain" may be either contiguous or non-contiguous. A binding domain must include at least 1 amino acid, and may include 2 or more, preferably 4 or more, amino acids which are contiguous or non-contiguous, but are necessary for modification-dependent binding to the binding partner, and may include a full-length protein. A binding domain of use in the invention may be present on a polypeptide chain that consists solely of the binding domain amino acid sequence or may be present in the context of a larger polypeptide molecule (i.e., one which comprises amino acids other than those of the binding domain), which molecule may be either naturally-occurring or recombinant and, in the case of the latter, may comprise either natural or non-natural amino acid sequences. As used herein, the term "engineered binding domain" refers to a binding domain, as defined above, which is an amino acid sequence that is altered (i.e., by insertion, deletion or substitution of at least one amino acid) such that the domain amino acid sequence is no longer as found in nature. The position of the altered amino acid is within the residues which form the domain.
An engineered binding domain of use in the invention may be present on a polypeptide chain that consists solely of the engineered binding domain amino acid sequence or may be present in the context of a larger polypeptide molecule (i.e., one which comprises amino acids other than those of the engineered binding domain), which molecule may be either naturally-occurring or recombinant and, in the case of the latter, may comprise either natural or non-natural amino acid sequences.
In an enzymatic assay of the invention, sites for post-translational modification may be present in either or both of the engineered domain and its binding partner, as defined above. If such sites are present on both the engineered domain and its binding partner, binding between the domain and its partner may be dependent upon the modification state of either one or both sites. If a single polypeptide chain comprises the engineered domain and its binding partner (or two engineered binding domains), the state of modification of one or both sites will determine whether binding between the two domains occurs.
As used herein with regard to modification of a polypeptide, the terms "site" and "site sufficient for the addition of refer to an amino acid sequence which is recognized by (i.e., a signal for) a modifying enzyme for the purpose of post-translational modification (i.e., addition or removal of a "moiety" as defined below) of the polypeptide or a portion thereof. A "site" additionally refers to the single amino acid which is modified. It is contemplated that a site comprises a small number of amino acids, as few as one but typically from 2 to 10, less often up to 30 amino acids, and further that a site comprises fewer than the total number of amino acids present in the polypeptide. In an enzymatic assay of the invention, a "site", for post-translational modification is present on an engineered binding domain and, optionally, its' binding partner, as defined below. If such sites are present on both engineered binding domain and the binding partner, binding between them may be dependent upon the modification state of either one or both sites. If a single polypeptide chain comprises the engineered binding domain and its binding partner (or two engineered binding domains), the state of post-translational modification of one or both sites will determine whether binding between the engineered binding domain and the binding partner occurs.
As used herein, the term "modification" or "post-translational modification" refers to the addition or removal of a chemical "moiety", as described herein, to/from a site on a polypeptide chain and does not refer to other post-translational events which do not involve addition or removal of such a moiety as described herein, and thus does not include simple cleavage of the reporter molecule polypeptide backbone by hydrolysis of a peptide bond, but does include hydrolysis of an isopeptide bond (e.g., in the removal of ubiquitin). As used interchangeably herein, the terms "moiety" and "group" refer to one of the post-translationally added or removed groups referred to herein: i.e., one of a phosphate, ubiquitin, glycosyl, fatty acyl, sentrin or ADP-ribosyl moiety.
As used herein, the term "binding partner" refers to a polypeptide or fragment thereof (a peptide) that binds to a binding domain, as defined herein, in a manner which is dependent upon the state of modification of a site for post-translational modification which is, at a minimum, present upon the binding domain; the binding partner itself may, optionally, comprise such a site and binding between the binding domain and its corresponding binding partner may, optionally, depend upon modification of that site. A binding partner does not necessarily have to contain a site for post-translational modification if such a site is not required to be present on it for modification-dependent association between it and a binding domain.
As used herein in reference to an engineered binding domain or other polypeptide, the term "isolated" refers to a molecule or population of molecules that is substantially pure (i.e., free of contaminating molecules of unlike amino acid sequence). As used herein, the terms "polypeptide"and "peptide" refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. D Polypeptide D refers to either a full-length naturally-occurring amino acid chain or a "fragment thereof or "peptide", such as a selected region of the polypeptide that is of interest in a binding assay and for which a binding partner is known or determinable, or to an amino acid polymer, or a fragment or peptide thereof, which is partially or wholly non-natural. ""Fragment thereof thus refers to an amino acid sequence that is a portion of a full-length polypeptide. between about 8 and about 500 amino acids in length, preferably about 8 to about 300, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids in length. "Peptide" refers to a short amino acid sequence that is 10-40 amino acids long, preferably 10-35 amino acids. Additionally, unnatural amino acids, for example,β-alanine, phenyl glycine and homoarginine may be included. Commonly- encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L- optical isomer. The L-isomers are preferred. In addition, other peptidomimetics are also useful, e.g. in linker sequences of polypeptides of the present invention (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267).
The term "synthetic", as used herein, is defined as that which is produced by in vitro chemical.
As used herein, the terms "protein", "subunit" and "domain" refer to a linear sequence of amino acids which exhibits biological function. This linear sequence includes full-length amino acid sequences (e.g. those encoded by a full-length gene or polynucleotide), or a portion or fragment thereof, provided the biological function, as well as the post-translational- modification-dependent binding function, is maintained by that portion or fragment. The terms subunit and domain also may refer to polypeptides and peptides having biological function. A peptide useful in the invention will at least have a binding capability, i.e, with respect to binding as or to a binding partner, and also may have another biological function that is a biological function of a protein or domain from which the peptide sequence is derived.
"Polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases in length and up to 1 ,000 bases or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. As used herein in reference to the purity of a molecule or population thereof, the term "substantially" refers to that which is at least 50%, preferably 60-75%, more preferably from 80-95% and, most preferably, from 98-100% pure.
As used herein in reference to a polypeptide, the term "engineered" refers to an amino acid sequence that is altered with respect to a natural amino acid sequence and particularly with respect to amino acids which contribute to modification-dependent binding of the polypeptide to a binding partner.
"Naturally-occurring" as used herein, as applied to a polypeptide or polynucleotide, refers to the fact that the polypeptide or polynucleotide can be found in nature. One such example is a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature. Once the polypeptide is engineered as described herein so as to associate with a binding partner in a modification-dependent manner where it did not formerly do so or where it did so in a manner different, either in degree or kind, from that which it was engineered to do, it is no longer naturally-ocurring but is derived from a naturally ocurring polypeptide.
In an assay of the invention, post-translational modification is reversible, such that a repeating cycles of addition and removal of a modifying moiety may be observed, although such cycles may not occur in a living cell found in nature.
As used herein, the term "associates" or "binds" refers to a polypeptide as described herein and its binding partner having a binding constant sufficiently strong to allow detection of binding by fluorescent or other detection means, which are in physical contact with each other and have a dissociation constant (Kd) of about lOμM or lower. The contact region may include all or parts of the two molecules. Therefore, the terms "substantially dissociated" and "dissociated" or "substantially unbound" or "unbound" refer to the absence or loss of contact between such regions, such that the binding constant is reduced by an amount which produces a discernable change in a signal compared to the bound state, including a total absence or loss of contact, such that the proteins are completely separated, as well as a partial absence or loss of contact, so that the body of the proteins are no longer in close proximity to each other but may still be tethered together or otherwise loosely attached, and thus have a dissociation constant greater than lOμM (Kd). In many cases, the Kd will be in the mM range. The terms "complex" and, particularly, "dimer", "multimer" and "oligomer"as used herein, refer to the engineered binding domain and its binding partner in the associated or bound state. More than one molecule of each of engineered binding domain and its binding partner may be present in a complex, dimer, multimer or oligomer according to the methods of the invention. As used herein, the term "binding sequence" refers to that portion of a polypeptide comprising at least 1, but also 2 or more, preferably 4 or more, and up to 8, 10, 100 or 1000 contiguous (i.e., covalently linked by peptide bonds) amino acid residues or even as many contiguous residues as are comprised by a full-length protein, that are sufficient for modification-dependent binding to a binding partner. A binding sequence may exist on a polypeptide molecule that consists solely of binding sequence amino acid residues or may, instead, be found in the context of a larger polypeptide chain (i.e., one that comprises amino acids other than those of the binding sequence).
As used herein, the term "engineered binding sequence" refers to a binding sequence, as defined above, that is altered (e.g., by insertion, deletion or substitution of at least one amino acid) such that the fragment amino acid sequence is no longer as found in nature. As in the case of an "engineered binding domain", as defined above, the alteration must occur in those amino acids of a polypeptide which contribute to modification-state-dependent binding (that is, within the binding domain).
As used herein, the term "binding polypeptide" refers to a molecule comprising multiple binding sequences, as defined above, which sequences are derived from a single, naturally-occurring polypeptide molecule and are both necessary and, in combination, sufficient to permit modification-state-dependent binding of the binding polypeptide to its binding partner, as defined above, wherein the sequences of the binding polypeptide are either contiguous or are non-contiguous. As used herein in reference to the component binding sequences of a binding polypeptide, the term "non-contiguous" refers to binding sequences which are linked by intervening naturally-occurring, as defined herein, or non-natural amino acid sequences or other chemical or biological linker molecules such are known in the art. The amino acids of a polypeptide that do not significantly contribute to the modification- state-dependent binding of that polypeptide to its binding partner may be those amino acids which are naturally present and link the binding sequences in a binding polypeptide or they may be derived from a different natural polypeptide or may be wholly non-natural. In assays of the invention, a binding polypeptide and its binding partner (which may, itself, be a binding domain, sequence or polypeptide, as defined herein) may exist on two different polypeptide chains or on a single polypeptide chain. As used herein, the term "engineered binding polypeptide" refers to a binding polypeptide. as defined above, which polypeptide comprises at least one engineered binding sequence, as described above. If the binding sequences of an engineered binding polypeptide are linked by amino acid sequences (rather than chemical or other non-peptide linkers), a naturally-occurring amino acid sequence which links binding fragments in a binding polypeptide of use in an assay of the invention may be derived from the same natural polypeptide sequence from which one or more of the component binding fragments are drawn, including that from which an engineered binding fragment may have been derived, or may instead be derived from a different natural polypeptide. It is preferred that in an isolated engineered binding domain and a binding partner therefor of the invention, the site comprises a sequence which directs modification by one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP- ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
It is additionally preferred that the site permits addition of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the addition prevents binding of the isolated engineered binding domain to the binding partner.
As used herein the term "prevents binding" or "prevents association" refers to the ability of at least one of the following: phosphate, ubiquitin, glycosyl, fatty acyl, sentrin or ADP-ribosyl group to inhibit the association, as defined above, of an isolated engineered binding domain and a binding partner thereof by at least 10%, preferably by 25-50%), highly preferably by 75-90%) and, most preferably, by 95-100% relative the association observed in the absence of such a modification under the same experimental conditions.
According to another preferred embodiment, the site permits addition of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP- ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the addition promotes binding of the isolated engineered binding domain to the binding partner. As used herein, the term "promotes binding" refers to that which causes an increase in binding of the engineered binding domain and its binding partner of at least two-fold, preferably 10- to 20-fold, highly preferably 50- to 100-fold, more preferably from 200- to 1000-fold, and, most preferably, from 200 to 10,000-fold. Preferably, the site permits removal of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the removal prevents binding of the isolated engineered binding domain to the binding partner.
In another preferred embodiment, the site permits removal of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and the removal promotes binding of the isolated engineered binding domain to the binding partner.
It is preferred that at least one of the isolated engineered binding domain and the binding partner comprises a detectable label, more preferred that the detectable label emits light and most preferred that the light is fluorescent.
A "fluorescent tag", "fluorescent label" or "fluorescent group" refers to either a fluorophore or a fluorescent protein or fluorescent fragment thereof. "Fluorescent protein" refers to any protein which fluoresces when excited with appropriate electromagnetic radiation. This includes proteins whose amino acid sequences are either natural or engineered. A "fluorescent protein" is a full-length fluorescent protein or fluorescent fragment thereof . By the same token, the term "linker" refers to that which is coupled to both the donor and acceptor protein molecules, such as an amino acid sequence joining two engineered binding domains, sequences or polypeptides or joining an engineered binding domain, sequence or polypeptide and its corresponding binding partner, or a disulfide bond between two polypeptide sequences, whether the sequences are present on the same- or on different polypeptide chains.
It is contemplated that with regard to fluorescent labels employed in FRET, the reporter labels are chosen such that the emission wavelength spectrum of one (the "donor") is within the excitation wavelength spectrum of the other (the "acceptor"). With regard to a fluorescent label and a quencher employed in a single-label detection procedure in an assay of the invention, it is additionally contemplated that the fluorophore and quencher are chosen such that the emission wavelength spectrum of the fluorophore is within the absorption spectrum of the quencher such that when the fluorophore and the quencher with which it is employed are brought into close proximity by binding of the engineered binding domain, sequence or polypeptide upon which one is present with the binding partner comprising the other, detection of the fluorescent signal emitted by the fluorophore is reduced by at least 10%, preferably 20-50%, more preferably 70-90% and. most preferably, by 95-100%. A typical quencher reduces detection of a fluorescent signal by approximately 80%>.
According to one preferred embodiment, one of the isolated engineered binding domain and the binding partner comprises a quencher for the detectable label.
The invention additionally provides a kit comprising an isolated engineered binding domain and a binding partner therefor, wherein the isolated engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site, and packaging materials therefor.
It is preferred' that the kit further comprises a buffer which permits modification- dependent binding of the isolated engineered binding domain and the binding partner. As used herein, the term "buffer" refers to a medium which permits activity of the protein-modifying enzyme used in an assay of the invention, and is typically a low-ionic- strength buffer or other biocompatible solution (e.g., water, containing one or more of physiological salt, such as simple saline, and/or a weak buffer, such as Tris or phosphate, or others as described hereinbelow), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate. Such a buffer permits dimerization of a non- phosphorylated and/or non-ubiquitinated and/or non-prenylated and/or non-sentrinated and/or non-ADP-ribosylated and/or non-glycosylated engineered binding domain of the invention and a binding partner therefor and, preferably, inhibits degradation and maintains biological activity of the reaction components. Inhibitors of degradation, such as protease inhibitors (e.g., pepstatin, leupeptin, etc.) and nuclease inhibitors (e.g., DEPC) are well known in the art. Lastly, an appropriate buffer may comprise a stabilizing substance such as glycerol, sucrose or polyethylene glycol.
As used herein, the term "physiological buffer" refers to a liquid medium that mimics the salt balance and pH of the cytoplasm of a cell or of the extracellular milieu, such that post-translational protein modification reactions and protei protein binding are permitted to occur in the buffer as they would in vivo. Preferably, the buffer additionally permits modification of the site for protein modification by one or more of the following enzymes: a kinase. a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
It is preferred that the kit further comprises one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine- Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase. It is additionally preferred that the kit further comprises a substrate for said enzyme which may be: MgATP, ubiquitin, sentrin, nicotinamide adenine dinucleotide (NAD+), uridine-diphosphate-N-acetylglucosamine-dolichyl-phosphate (UDP-N-acetylglucosamine- dolichyl-phosphate), palmytyl CoA, myristoyl CoA and UDP-N-acetylglucosamine.
It is contemplated that at least a part of a substrate of an enzyme of use in the invention is transferred to an modification site on an isolated engineered binding domain of the invention. As used herein, the term "at least a part of a substrate" refers to a portion (e.g., a fragment of an amino acid sequence, a moiety or a group, as defined above) which comprises less than the whole of the substrate for the enzyme, the transfer of which portion to a modification site on an isolated engineered binding domain and, optionally, to a site on a binding partner therefor, both as defined above, is catalyzed by the enzyme.
Preferably, the kit further comprises a cofactor for said enzyme. Cofactors of use in the invention include, but are not limited to, cAMP, phosphotidylserine, diolein, Mn2+ and Mg2+.
It is preferred that at least one of the isolated engineered binding domain and the binding partner comprises a detectable label, more preferred that the detectable label emits light and most preferred that the light is fluorescent. An enzyme of use in the invention may be natural or recombinant or, alternatively, may be chemically synthesized. If either natural or recombinant, it may be substantially pure
(i.e.. present in a population of molecules in which it is at least 50% homogeneous), partially purified (i.e.. represented by at least 1% of the molecules present in a fraction of a cellular lysate) or may be present in a crude biological sample.
As used herein, the term "sample" refers to a collection of inorganic, organic or biochemical molecules which is either found in nature (e.g., in a biological- or other specimen) or in an artificially-constructed grouping, such as agents which might be found and/or mixed in a laboratory. Such a sample may be either heterogeneous or homogeneous. As used herein, the interchangeable terms "biological specimen" and "biological sample" refer to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. Lastly, "biological sample" refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
As used herein, the term "organism" refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
In a method as described above, it is preferred that at least one of the isolated engineered binding domain and the binding partner is labeled with a detectable label, more preferred that the label emits light and most preferred that the light is fluorescent. Preferably, the detection step is to detect a change in signal emission by the detectable label.
According to one preferred embodiment, the method further comprises exciting the detectable label and monitoring fluorescence emission.
Preferably, the enzyme is one of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
It is preferred that the method further comprises the step, prior to or after the detection step, of contacting the isolated engineered binding domain and the binding partner with an agent which modulates the activity of the enzyme.
As used herein with regard to a biological or chemical agent, the term "modulate" refers to enhancing or inhibiting the activity of a protein-modifying enzyme in an assay of the invention; such modulation may be direct (e.g. including, but not limited to, cleavage of- or competitive binding of another substance to the enzyme) or indirect (e.g. by blocking the initial production or, if required, activation of the modifying enzyme).
"Modulation" refers to the capacity to either increase or decease a measurable functional property of biological activity or process (e.g., enzyme activity or receptor binding) by at least 10%, 15%, 20%>, 25%, 50%, 100%o or more; such increase or decrease may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
The term "modulator" refers to a chemical compound (naturally occurring or non- naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non- peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule. Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein. The activities (or activity) of a modulator may be known, unknown or partially-known. Such modulators can be screened using the methods described herein.
The term "candidate modulator" refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator. Usually, various predetermined concentrations are used for screening such as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM, as described more fully hereinbelow. Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target. The invention also provides a method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El. a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase, the method comprising contacting an isolated engineered binding domain, a binding partner therefor and an enzyme with a candidate modulator of the enzyme, wherein the engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner that is dependent upon modification of the site by the enzyme, and wherein at least one of the isolated engineered binding domain and the binding partner comprises a detectable label, and monitoring the binding of the isolated engineered binding domain to the binding partner, wherein binding or dissociation of the isolated engineered binding domain and the binding partner as a result of the contacting is indicative of modulation of enzyme activity by the candidate modulator of said enzyme.
It is preferred that the detectable label emits light and highly preferred that the light is fluorescent.
Preferably, the monitoring comprises measuring a change in energy transfer between a label present on the isolated engineered binding domain and a label present on the binding partner.
The invention also provides a method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N- acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase, the method comprising contacting an assay system with a candidate modulator of enzymatic activity of such an enzyme, and monitoring binding of an isolated engineered binding domain and a binding partner therefor in the assay system, wherein the engineered binding domain includes a site for post-translational modification and binds the binding partner in a manner that is dependent upon modification of the site by at least one such enzyme in the assay system, wherein at least one of the isolated engineered binding domain and the binding partner comprises a detectable label, and wherein binding or dissociation of the isolated engineered binding domain and the binding partner as a result of the contacting is indicative of modulation of enzyme activity by the candidate modulator of such an enzyme.
In a particularly preferred embodiment, in one of the methods described above, the method comprises real-time observation of association of an isolated engineered binding domain and its binding partner.
As used herein in reference to monitoring, measurements or observations in assays of the invention, the term "real-time" refers to that which is performed contemporaneously with the monitored, measured or observed events and which yields a result of the monitoring, measurement or observation to one who performs it simultaneously, or effectively so, with the occurrence of a monitored, measured or observed event. Thus, a "real time" assay or measurement contains not only the measured and quantitated result, such as fluorescence, but expresses this in real time, that is, in hours, minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shorter times exceed the instrumentation capability; further, resolution is also limited by the folding and binding kinetics of polypeptides.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.
BRIEF DESCRIPTIONS OF THE DRAWINGS
Figure 1 depicts double- and single-chain enzymatic assay formats of the invention. Figure 2 presents a schematic overview of FRET in an assay of the invention. Figure 3 presents monomer: excimer fluorescence.
Figure 4 presents sUb-N protein and sUb-C phage interaction monitored by surface plasmon resonance (SPR).
Figure 5 presents the detection of bound ZAPGFP protein by GFP fluorescence. DESCRIPTION
The invention is based upon the discovery that a binding polypeptide or a polypeptide comprising a binding domain or sequence, each as defined herein, which binding polypeptide, domain or sequence has been engineered so as to associate with a binding partner in a manner that is dependent upon the presence or absence of a "moiety", as described herein, at a site for post-translational modification on the same polypeptide chain provides a sensitive system for the activity of an enzyme that catalyzes post-translational protein modification at such a site and that measurements of enzymatic activity performed in such a system may be taken in real time.
A. Engineered binding polypeptides. sequences and domains of use in the invention
An assay of the invention utilizes at least one polypeptide chain which comprises a sequence that has been engineered to associate specifically with a second sequence, or "binding partner" as defined herein, in a modification-dependent manner.
i. Post-translational protein modifications in the invention
ADP-ribosylation
Mono-ADP-ribosylation is a post-translational modification of proteins which is currently thought to play a fundamental role in cellular signalling. A number of mono-ADP- ribosyl-transferases have been identified, including endogenous enzymes from both bacterial and eukaryotic sources and bacterial toxins. A mono-ADP-riboylating enzyme, using as substrates the protein to be modified and nicotinamide adenine dinucleotide (NAD+), is NAD:Arginine ADP ribosyltransferase (Zolkiewska et al, 1992, Proc. Natl. Acad. Sci. U.S.A., 89: 11352-11356). The reactions catalyzed by bacterial toxins such as cholera and pertussis toxin are well understood; the activities of these toxins result in the permanent modification of heterotrimeric G proteins. Endogenous transferases are also thought to modify G proteins and therefore to play a role in signal transduction in the cell (de Murcia et al., 1995, Trends Cell Biol. 5: 78-81). The extent of the effects that ADP-ribosylation can mediate in the cell is illustrated by the example of brefeldin A, a fungal toxin metabolite of palmitic acid. This toxin induces the mono-ADP-ribosylation of BARS-50 (a G protein involved in membrane transport) and glyceraldehyde-3 -phosphate dehydrogenase. The cellular effects of brefeldin A include the blocking of constitutive protein secretion and the extensive disruption of the Golgi apparatus. Inhibitors of the brefeldin A mono-ADP-ribosyl- transferase reaction have been shown to antagonise the disassembly of the Golgi apparatus induced by the toxin (Weigert et al., 1997. J. Biol. Chem., 272: 14200-14207). A number of amino acid residues within proteins have been shown to function as ADP-ribose acceptors. Bacterial transferases have been identified which modify arginine, asparagine, cysteine and diphthamide residues in target proteins. Endogenous eukaryotic transferases are known which also modify these amino acids, in addition there is evidence that serine, threonine, tyrosine, hydroxyproline and histidine residues may act as ADP-ribose acceptors but the relevant transferases have not yet been identified (Cervantes-Laurean et al., 1997, Methods Enzvmol., 280: 275-287 and references therein).
Poly-ADP-ribosylation is thought to play an important role in events such as DNA repair, replication, recombination and packaging and also in chromosome decondensation. The enzyme responsible for the poly-ADP-ribosylation of proteins involved in these processes is poly (ADP-ribose) polymerase (PARP; for Drosophila melanogaster PARP, see Genbank Accession Nos. D 13806, D 13807 and D 13808). The discovery of a leucine zipper in the self-poly(ADP-ribosyl)ation domain of the mammalian PARP (Uchida et al., 1993, Proc. Natl. Acad. Sci. U.S.A.. 90: 3481-3485) suggested that this region may be important for the dimerization of PARP and also its interaction with other proteins (Mendoza- Alvarez et al., 1993. J. Biol. Chem.. 268: 22575-22580).
Specific examples of ADP ADP-ribosylation sites are those found at Cys3 and Cys4 (underlined) of the B-50 protein (Coggins et al., 1993, J. Neurochem.. 60: 368-371; SwissProt Accession No. P06836): MLCCMRRTKQVE KNDDD and Pγ (the γ subunit of cycylic CMP phophodiesterase; Bondarenko et al., 1997, J. Biol. Chem.. 272: 15856-15864; Genbank Accession No. X04270): FKQRQTRQFK .
Ubiquitination Ubiquitination of a protein targets the protein for destruction by the proteosome. This process of destruction is very rapid (X.\ ~ 60 seconds), and many proteins with rapid turnover kinetics are destroyed via this route. These include cyclins, p53, transcription factors and transcription regulatory factors, among others. Thus, ubiquitination is important in processes such as cell cycle control, cell growth, inflammation, signal transduction; in addition, failure to ubiquitinate proteins in an appropriate manner is implicated in malignant transformation. Ubiquitin is a 76-amino-acid protein which is covalently attached to a target protein by an isopeptide bond, between the ε-amino group of a lysine residue and the C-terminal glycine residue of ubiquitin. Such modification is known as mono-ubiquitination, and this can occur on multiple Lys residues within a target protein. Once attached, the ubiquitin can itself be ubiquitinated, thus forming extended branched chains of polyubiquitin. It is this latter state which signals destruction of the target protein by the proteosome. In the process of destruction, it appears that the polyubiquitinated protein is taken to the proteosome via a molecular chaperone protein, the ubiquitin molecules are removed undamaged (and recycled) and the target is degraded.
Ubiquitination is a complex process, which requires the action of three enzymes: Ubiquitin activating enzyme El (for human, Genbank Accession No. X56976), ubiquitin conjugating enzyme E2, also referred to as the ubiquitin carrier protein, (for human 17kDa form, Genbank Accession No. X78140) and Ubiquitin protein ligase E3α (UBR1; human, Genbank Accession No. AF061556). There are multiple forms of each of these enzymes in the cell, and the above examples are, therefore, non-limiting.
The signals contained within a protein which determine whether the protein is subject to the process of ubiquitination and destruction are two-fold: first, the identity of the N- terminal amino acid (so called N-end rule, Varshavsky, 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 12142-12149), and secondly the presence of a suitably positioned Lys residue in the protein (Varshavsky, 1996, supra). This Lys can be up to -30 amino acids away from the N- terminus in experimental examples studied where the N-terminus is a flexible, poorly- structured element of the protein (Varshavsky, 1996, supra) or could potentially be anywhere in the sequence where this presents it at an appropriate location relative to the N-terminus.
An appropriate location is one which allows interaction of both the N-terminal residue and this integral lysine with the enzyme(s) responsible for ubiquitination, presumably simultaneously. The Lys residue becomes ubiquitinated, and the process of destruction is initiated. N-terminal residues can be classed as stabilizing (s) or destabilizing (d), and the inclusion of an amino acid in one of these broad classes is species-dependent (prokaryotes differ from yeast, which differs from mammals; Varshavsky, 1996, supra). In a dimeric (or other oligomeric protein) the destabilizing N-terminal residue and the internal Lys can be in cis (on a single peptide), but may also be in trans (on two different polypeptides). The trαrø'-recognition event will only take place while the complex is physically associated. Only the ubiquitinated subunit is proteolyzed (Varsharsky, 1996, supra).
Two examples of ubiquitination sites from natural proteins, IκB (Dai et al., 1998,_J. Biol. Chem, 273: 3562-3573; Genbank Accession No. M69043) and β-galactosidase (Johnson et al., 1990, Nature. 346: 287-291) are as follows:
IκB NH3 -MFQAAERPQE AMEGPRDGLKKERLLDDRH-COOH
β-galactosidase NH3 -HGSGAWLLPVSLVKRKTTLAP - COOH
where the ubiquitinated lysine residue is underlined for each (e.g., Lys*5 and Lysπ for β- galactosidase).
According to the invention, a ubiquitination assay measures the addition of ubiquitin to-, rather than the destruction of-, an engineered binding domain, sequence or polyeptpide.
Glycosylation N-linked glycosylation is a post-translational modification of proteins which occurs in the endoplasmic reticulum and golgi apparatus and is utilized with some proteins en route for secretion or destined for expression on the cell surface or in another organelle. The carbohydrate moiety is attached to Asn residues in the non-cytoplasmic domains of the target proteins, and the consensus sequence (Shakineshleman, 1996, Trends Glvcosci. Glycotech., 8: 115-130) for a glycosylation site is: NxS/T, where x cannot be proline or aspartic acid. N-linked sugars have a common five-residue core consisting of two GlcNAc residues and three mannose residues due to the biosynthetic pathway. This core is modified by a variety of Golgi enzymes to give three general classes of carbohydrate known as oligomarmosyl, hybrid and lactosamine-containing or complex structures (Zubay, 1998, Biochemistry, Wm. C. Brown Publishers). An enzyme known to mediate N-glycosylation at the initial step of synthesis of dolichyl-P-P-oligosaccharides is UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase (for mouse, Genbank Accession Nos. X65603 and S41875).
Oxygen-linked glycosylation also occurs in nature with the attachment of various sugar moieties to Ser or Thr residues (Hansen et al., 1995, Biochem. J.. 308: 801-813). Intracellular proteins are among the targets for O-glycosylation through the dynamic attachment and removal of O-N-Acetyl-D-glucosamine (O-GlcNAc; reviewed by Hart, 1997, Ann. Rev. Biochem., 66: 315-335). Proteins known to be O-glycosylated include cytoskeletal proteins, transcription factors, the nuclear pore protein complex, and tumor- suppressor proteins (Hart, 1997, supra). Frequently these proteins are also phosphoproteins, and there is a suggestion that O-GlcNAc and phosphorylation of a protein play reciprocal roles. Furthermore, it has been proposed that the glycosylation of an individual protein regulates proteimprotein interactions in which it is involved.
Specific sites for the addition of O-GlcNAc are found, for example, at Ser277, Ser3ι6 and Ser383 of p67SRF (Reason et al., 1992, J. Biol. Chem., 267: 16911-16921 ; Genbank Accession No. J03161). The recognition sequences encompassing these residues are shown below: 74GTTSTIQTAP 313SAVSSADGTVLK 374DSSTDLTQTSSSGTVTLP The identity of sites of O-GlcNAc is additionally known for a small number of proteins including c-myc (Thr58, also a phosphorylation site; Chou et al., 1995, J. Biol. Chem., 270: 18961-18965), the nucleopore protein p62 (see Reason et al., 1992, supra):
MAGGPADTSDPL and band 4.1 of the erythrocyte ( see Reason et al, 1992, supra): AQTITSETPSSTT.
The site at which modification occurs is, in each case, underlined. The reaction is mediated by O-GlcNAc transferase (Kreppel et al, 1997, J. Biol. Chem., 272: 9308-9315). These sequences are rich in helix breaking residues (e.g., G and P) and may be difficult to incorporate into a helical framework. Prenylation (fatty acylation)
The post-translational modification of proteins with fatty acids includes the attachment of myristic acid to the primary amino group of an N-terminal glycine residue (Johnson et al., 1994, Ann. Rev. Biochem., 63: 869-914) and the attachment of palmitic acid to cysteine residues (Milligan et al., 1995. Trends Biochem. Sci., 20: 181-186).
Fatty acylation of proteins is a dynamic post-translational modification which is critical for the biological activity of many proteins, as well as their interactions with other proteins and with membranes. Thus, for a large number of proteins, the location of the protein within a cell can be controlled by its state of prenvlation (fatty acid modification) as can its ability to interact with effector enzymes (ras and MAP kinase, Itoh et al., 1993, J. Biol. Chem., 268: 3025-; ras and adenylate cyclase (in yeast; Horiuchi et al., 1992, Mol. Cell. Biol., 12: 4515-) or with regulatory proteins (Shirataki et al., 1991, J. Biol. Chem., 266: 20672-20677). The prenylation status of ras is important for its oncogenic properties (Cox, 1995, Methods Enzymol.. 250: 105-121) thus interference with the prenylation status of ras is considered a valuable anti-cancer strategy (Hancock, 1993, Curr. Biol., 3: 770).
Sentrinization
Sentrin is a novel 101-amino acid protein which has 18 % identity and 48% similarity with human ubiquitin (Okura et al., 1996, J. Immunol., 157: 4277-4281). This protein is known by a number of other names including SUMO- 1 , UBL 1 , PIC 1 , GMP 1 and SMT3 C and is one of a number of ubiquitin-like proteins that have recently been identified. Sentrin is expressed in all tissues (as shown by Northern blot analysis), but mRNA levels are higher in the heart, skeletal muscle, testis, ovary and thymus.
The sentrinization of proteins is thought to involve the Ubiquitin-conjugating enzyme Ubc9 (Gong et al., 1997, J. Biol. Chem., 272: 28198-28201). The interaction between these two proteins in the yeast two-hybrid screen is very specific, suggesting that this is a biologically relevant phenomenon. The interaction is dependent upon the presence of the conserved C-terminal Gly-Gly residues present in sentrin (Gong et al., 1997, supra). The conjugation of sentrin to other proteins via Gly requires the cleavage of the C-terminal four amino acids of the protein, His-Ser-Thr-Val.
One important protein shown to be modified by the addition of sentrin is the Ran- specific GTPase-activating protein, RanGAPl, which is involved in nuclear import of proteins bearing nuclear-localization signals (Johnson and Hochstrasser, 1997, Trends Cell Biol.. 7: 408-413). Conjugation of RanGAPl and sentrin is essential both for the targeting of RanGAPl to its binding partner on the nuclear pore complex (NPC) and for the nuclear import of proteins. Sentrin itself does not bind with high affinity to the NPC and it is, therefore, likely that it either provokes a conformational change in RanGAP 1 that exposes a binding site or, alternatively, that the binding site is formed using both sentrin and RanGAPl sequences. There is evidence to suggest that the conjugation of sentrin to RanGAPl is necessary for the formation of other sentrinized proteins (Kamitani et al., 1997, J. Biol. Chem.. 272: 14001-14004) and that the majority of these sentrinized proteins are found in the nucleus.
Sentrin has been shown in yeast two-hybrid screens to interact with a number of other proteins, including the death domains of Fas/APOl and the TNF receptors, PML, RAD51 and RAD52 (Johnson and Hochstrasser, 1997, supra). These interactions implicate sentrin in a number of important processes. Fas/APOl and TNF receptors are involved in transducing the apoptosis signal via their death domains. Ligation of Fas on the cell surface results in the formation of a complex via death domains and death-effector domains, triggering the induction of apoptosis. The overexpression of sentrin protects cells from both anti-Fas/ APO and TNF-induced cell death (Okura et al., 1996, supra). It is not clear whether this protection is achieved simply by preventing the binding of other proteins to these death domains or whether a more complex process is involved, possibly one involving the ubiquitin pathway.
The interaction of sentrin with PML (a RING finger protein) is important, as it points to a disease state in which this protein may play a role. In normal myeloid cells, PML is found in a nuclear multiprotein complex known as a nuclear body. These nuclear bodies are disrupted in acute promyelocytic leukaemia, where a chromosomal translocation generates a fusion between regions of the retinoic acid receptor α and PML. This disruption can be reversed by treatment with retinoic acid. It has been shown that PML is covalently modified at multiple sites by members of the sentrin family of proteins (but not by ubiquitin or NEDD8). Two forms of the aberrant fusion protein have been identified, neither of which is modified by sentrin. It is, therefore, thought that differential sentrinization of the normal and aberrant forms of PML may be important in the processes underlying acute promyelocytic leukaemia and may help in the understanding of the biological role of the PML protein (Kamitani et al., 1998. J. Biol. Chem.. 273: 3117-3120). Phosphorylation - kinase and phosphatases A particularly important post-translational modification for which a large number of enzymes and targets have been identified is phoshorylation and dephosphorylation. The art is replete with references to said enzymes, i.e. protein kinases and phosphatases, and their targets, including consensus phosphorylation motifs (such as -SQ- or -TQ- for the DNA dependent protein kinase (DNA-PK).
Some non-limiting examples of kinases and their sites for post-translational modification are presented in Table 2 below (phosphorylation/dephosphorylation)
Further examples of protein kinases identified to date include the protein tyrosine kinase subfamily (such as PDGF receptors, EGF receptors, src family kinases (see Brown and Cooper, 1996, Biochimica and Biophysica Acta 1287: 121-149 for a review), the JAK kinase family (such as JAKl , JAK2 and tyk2), Erb B2, Bcr-Abl, Alk, Trk, Res/Sky - for a detailed review see Al-Obeidi et al, 1998, Biopolymers (Peptide Science), Vol 47: 197-223), the MAP kinase pathway subfamily (such as the MAP family, the ERK family, the MEK family, the MEKK family, RAF-1 and JNK), the cyclin-dependent kinase subfamily (such as p34c c and cdk2 - see Nigg, 1995, Bioessays 17: 471-480 for a review), Weel/Mytl, polo-like kinases (such as plkl, Plxl, POLO, Snk, Fnk/Prk Sak-a, Sak-b - see Lane and Nigg, 1997, Trends in Cell Biol. 7: 63-68), the receptor serine kinase subfamily, protein kinase C (PK-C), cyclic-AMP dependent kinase (PK-A), cyclic-GMP dependent kinase, Ca2+/calmodulin dependent kinases (such as CaM kinase I, II and IV), DNA dependent protein kinase,), phosphoinositide 3 -kinases, PDK-1, the p21 -activated protein kinase family (PAKs), such as Pakl, Pak2 and Pak3- see Sells and Chernoff, 1997, Trends in Cell Biol. 7: 162-167), p70 S6 kinase, IkB kinase, casein kinase II, glycogen-synthase kinases.
A discussion of particular kinase pathways involved in signal transduction is given in chapter 35 of Lewin, 1997, Gene VI, Oxford University Press.
Details of recognition and binding domains for a variety of kinases are given in Kuriyan and Cowburn, 1997, Annu. Rev. Biophys. Biomol. Struc. 26:259-288. Some specific examples of kinases whose activity may be studied using the methods of the invention include the src family tyrosine kinases Lck and Fyn, that phosphorylate the TCR ζ chain, and are known to be involved in signal transduction associated with T cell receptor stimulation. The TCR ζ chain comprises specific tyrosine residues present in immunoreceptor tyrosine-based activation motifs (ITAMs) that are phosphorylatd by Lck and Fyn (Kuriyan and Cowburn, 1997, ibid.). The SH2 domain of another tyrosine kinase, ZAP70 binds to phosphorylated TCR ζ. Thus TCR ζ ITAM and ZAP70 SH2 represent binding domains and binding partners that may be of interest in studying the activity of the kinases Lck and Fyn (see Elder et al., 1994, Science 264: 1596-1599 and Chan et al., 1994, Science 264: 1599- 1601.
Another example is the IgE receptor γ subunit and the SH2 domain of Syk that may be used to study the activity of the Lyn kinase.
Examples of phosphatase identified to date fall into three main families (for review see Barford et al., 1998, Annu. Rev. Biophys. Biomol. Struc. 27: 133-164). The PPP family includes the following catalytic subunits: PPlc, PP2Ac, PP2B, PPP1, PPP2A and PPP5 and the following regulatory subunits: NIPP-1, RIPP-1, p53BP2, γ,34.5, PR65, PR55, PR72, PTPA, SV40 small T antigen, PPY, PP4, PP6 and PP5.
The PPM family includes pyruvate dehydrogenase phosphatase and Arabidopsis ABU .
The protein tyrosine phosphatase family includes PTP1B, SHP-1, SHP-2 (cytosolic non- receptor forms), CD45 (see Thomas and Brown, 1999, Trends in Immunol, 20: 406 and Ashwell and D'Oro, 1999, Trends in Immunol, 20: 412 for further details), RPTP (receptorlike, transmembrane forms) and cdc25, kinase-associated phosphatase and MAP kinase phosphatase- 1 (dual-specificity phosphatases). PTP1B is known to associate with the insulin receptor in vivo (Bandyopadhyay et al., 1997, J. Biol. Chem. 272: 1639-1645). ii. Assay of enzymatic activity according to the invention
An assay of the invention may be performed using either a single-chain or double- chain format, as illustrated in Figure 1. In Figure 1 , an engineered binding domain may associate with a second amino acid sequence (or "binding partner") present on the same polypeptide chain or, alternatively, with a binding partner present on a second polyepeptide chain. The binding partner may, itself, be an engineered binding polypeptide (i.e., one which is engineered so as to be dependent upon the presence or absence of a chemical moiety at a site for post-translational modification in order to participate in proteimprotein binding) or may be a natural sequence or a non-natural amino acid sequence which does not comprise a site for post-translational modification that affects proteimprotein binding. The complex between an engineered binding domain and a binding partner may, therefore, comprise a self- associated polypeptide monomer or. alternatively, either a hetero- or homo-oligomer.
Non-limiting examples of pairs of amino acid sequences which associate in nature and can be engineered to provide engineered binding domains, sequences or polypeptides of use in the invention are presented in Table 1. One or, alternatively, both members of a pair can be engineered to comprise sites for post-translational modification.
Table 1
Ron and Mochlv- Rosen, 1995, Proc. Natl. Acad. Sci. U.S.A., 92: 492-496.
The association of these two regions will have a number of determinants other than the engineered chemical modification in vivo including the presence or absence of PKC activators (such as phosphatidylserine, Ca2+ and diacylglycerol) or endogenous RACK1. This might complicate interpretation of the assay in vivo, however it should provide a useful module on which to build an in vitro assay.
In order for the SH2 domain to be useful in an assay of this type it must be modified such that the addition or removal of a phosphate group from a tyrosine residue is no longer a determinant of binding. This could be achieved by thiophosphorylation of the Tyr residue in an in vitro assay to yield a permanently phosphorylated protein. Alternatively, it may be possible to mimic phosphorylation by the mutuation of the key Tyr residue to Glu or Asp. If this were possible then these domains could be used in an in vivo assay. ** Wang et al... 1997, J. Biol. Chem.. 272: 17542-17550.
Konishi et al.. 1994, Biochem. Biophys. Res. Comm., 205: 1770-1775. a Fushman et al., 1998, J. Biol. Chem.. 273: 2835-2843. φ Again, in order for the PTB domain to be useful in an assay of this type it must be modified such that the addition or removal of a phosphate group from a yrosine residue is no longer a determinant of binding. This could be achieved by thiophosphorylation of the Tyr residue in an in vitro assay to assay as it is not clear whether any of the proteins bind in a competitive manner.yield a permanently phosphorylated protein. Alternatively, it may be possible to mimic phosphorylation by the mutation of the key Tyr residue to Glu or Asp. If this were possible then these domains could be used in an in vivo assay. ς Klauck et al., 1996, Science. 271: 1589-92.
Nauert et al., 1997, Curr. Biol.. 7: 52-62.
* Puls et al.. 1997. Proc. Natl. Acad. Sci. U.S.A.. 94: 6191-6196.
ZIP contains more than one protein binding motif (YXDED motif, ZZ zinc finger) and is known to bind to several proteins other than PDC ζ (including p62 and EBIAP) and also to self-associate (this self association is in competition with PKC ζ binding). These multiple interactions may cause problems with an in vivo assay as it is not clear whether any of the proteins bind in a competitive manner.
Sites for post-translational modification may be selected according to the specificity of enzyme(s) to be assayed. Non-limiting examples of sites for post-translational modification are presented in Tables 2 (phosphorylation/dephosphorylation) and 3 (addition/removal of other chemical moieties). Table 2
X signifies any amino acid.
Consensus sequences are taken from Trends Biochem. Sci. (1990) 15: 342-346. Further examples are tabulated in Pearson and Kemp, 1991. Methods Enzvmol., 200: 62-81.
A large number of assays can be conceived, based upon the principles outlined above. The principle can be summarized, in this case for phosphorylation, as follows: (Fl-E-partnerl) (F2-partner2) + ATP → Fl-EP-partnerl + F2-partner2 + ADP FRET No FRET
The following alternative assay format also is envisaged:
Fl-E-partnerl + F2-partner2 + ATP → (Fl-EP-partnerl) (F2-partner2) + ADP No FRET FRET
Where E = engineered phosphorylation site P = phosphorylation FI = donor fluorophore F2 = acceptor fluorophore FRET = Fluorescent resonance energy transfer
Table 3
A simple FRET assay based upon these modifications to site for post-translational modification present on an engineered binding domain, sequence or polypeptide may be performed as presented below. It is contemplated that other light-based detection assays, such as those involving single labels, labels and corresponding quenchers, etc. can be employed.
(Fl-E-partnerl)(F2-partner2) + substrate → Fl-EM-partnerl + F2-partner2 + byproduct (FRET) (No FRET)
where: E = engineered modification site
M = modification F 1 = donor fluorophore F2 = acceptor fluorophore
-33- Alternatively, a FRET-based assay may follow a format such as:
Fl-E-partner + F2-partner + substrate → (Fl-EM-partnerl)(F2-partner2) + byproduct (No FRET) (FRET)
Placement of the modification site may be determined empirically (see below), such that the location itself permits the interaction between the engineered binding domain, sequence or polypeptide and a binding partner but that the association is altered on modification of the engineered site. This change in association may be a direct or indirect consequence of modification. While not being bound to any theory, such a change may be based on, for example, a conformational or electrostatic change brought about by phosphorylation or dephosphorylation. In cases where there is no appropriate structural information, the sites for the attachment of a fluorophore or other label or quencher will also be determined empirically. Table 4 lists enzymes which perform the several modifications discussed herein as being of use in the invention.
Table 4
iii. Design of engineered binding domains and binding partners therefor for use in the invention
According to the invention, a binding domain is engineered such that its association with a binding partner is dependent upon post-translational modification at a site for post- translational modification which is introduced into- or altered within- the binding domain. The binding domain which undergoes engineering may, itself, be a naturally-occurring amino acid sequence or may be non-natural. Such engineering is performed by molecular methods which are well known in the art, as described below. The location of the engineered post- translational modification site must be such that it is tolerated in one state of modification (for example, prior to modification), but provokes dissociation of the complex in the opposite state of modification (following modification; or vice versa). As stated above, placement of the modification site within the domain and, optionally, the corresponding binding partner may involve empirical testing on a case-by-case basis; however, such testing can be facilitated through the use of knowledge of the structural basis of the interaction sites in the complex. Such knowledge may be structural (e.g., using crystallographic data or a molecular modeling algorithm of the 3 -dimensional structure of the protein or proteins involved in the complex of interest), a functional assessment of the regions of primary sequence important in binding, or a combination of these. These data will identify regions of the protein most likely to be influenced by the insertion of a post-translational modification site.
The contact face between components of the complex is one location at which a site for post-translational modification might be engineered, but it is not the only useful location.
The modification of a site remote from the interface site(s) can also lead to binding or dissociation of the complex. This would be expected to occur upon long-range alterations in protein structure as a consequence of the post-translational modification, which could be as extreme, for example, as structural collapse following modification.
A peptide, PKI(5-24amide), derived from a protein inhibitor of the cAMP-dependent protein kinase binds to the active site of protein kinase A (PKA) with high affinity. The 3-D structure of this complex is known (Knighton et al., 1991, Science, 253: 414-420) as is a functional dissection of the sequence of this peptide to identify residues involved in this biological activity (Glass et al., 1989, J. Biol. Chem., 264: 8802-8810). The binding of PKI(5-24amide) to the catalytic subunit of PKA can be monitored by a number of techniques including FRET, fluorescence correlation spectroscopy (FCS) or fluorescence anisotropy provided both components in the former case or the PKI(5-24amide) component in the latter two cases, respectively, are labeled with appropriate fluorophores.
The introduction of a PKA phosphorylation site into this peptide, by mutation of 1
Ala Ser (called PKI(A21S) hereinafter), results in a reporter molecule for protein kinase A activity. When used in an assay of the invention, PKI(A21S) binds to PKA when dephosphorylated, but dissociates from the enzyme once phosphorylated.
In some instances the binding partner might require mutation of its primary sequence to accommodate the post-translational modification site introduced into the engineered binding domain.
Molecular methods useful in producing an engineered binding domain
An engineered binding domain of use in the invention is produced using molecular methods such as are known in the art (see, for example, Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al, Current Protocols in Molecular Biology, copyright 1987- 1994, Current Protocols, copyright 1994-1998, John Wiley & Sons, Inc.). Such methods include chemical synthesis of a polyeptide sequence that encompasses an engineered binding domain or expression of a recombinant polynucleotide encoding such a molecule. Such a polynucleotide may be chemically synthesized; however, of particular use in the invention are methods of in vitro or otherwise site-directed mutagenesis by which to engineer a site for post-translational modification into an existing binding domain (whether natural or previously engineered) or by which to alter the enzyme specificity of an existing site. Typically, methods for in vitro mutagenesis comprise the annealing of a mutagenic oligonucleotide primer comprising the desired alteration to a complementary, single-stranded template, followed by second strand synthesis, whether using single-cycle synthesis or polymerase chain reaction (PCR). Cloning and sequencing are then performed to identify and isolate molecules bearing the desired alterations. Such mutagenesis methods optionally include a selection for mutated molecules, either through the use of modified nucleotides incorporated into the nascent polynucleotide strand or through the incorporation of a restriction site into the vector bearing the first strand which is disrupted in the second strand (i.e., in coupled priming; Carter et al., 1985, Nucleic Acids Res., 13: 4431-4443) and, with either technique, subsequent transformation of the first and second strands into a strain of host cells that selectively destroys the first strand and propagates the second.
Kits and individual components for in vitro mutagenesis enjoy wide commercial availability. A non-limiting sampling of such kits is as follows: From Stratagene (LaJolla, CA, U.S.A.): ExSite™ PCR-Based Site-Directed Mutagenesis Kit (catalog number: 200502)
QuikChange™ Site-Directed Mutagenesis Kit (catalog number: 200518) Chameleon™ Double-Stranded, Site-Directed Mutagenesis Kit (catalog numbers: 200508 and 200509 From Promega (Madison, WI, U.S.A.): Interchange™ in vivo Amber Suppressor Mutagenesis System (catalog number:
Q5080
Altered Sites® II in vitro Mutagenesis Systems (catalog numbers: Q6210, Q6090 and Q6080)
GeneEditor™ in vitro Site-Directed Mutagenesis System (catalog number: Q9280)
Erase-a-Base® System (catalog numbers E5850 and E 5750) From New England Biolabs (Beverly, MA, U.S.A.):
Code20™ Cassette Mutagenesis System (catalog number: 7520) All such kits are used according to the manufacturer's instructions. iv. Selection of functional partners sensitive to post-translation modification An engineered binding domain generated as described above may then be assayed for modification-dependent binding to a binding partner with which it was known to associate prior to engineering or; if binding of the the engineered binding domain and the binding partner is determined to be modification-sensitive (i.e., such that the engineered binding domain and the binding partner either do- or do not associate, depending upon modification of the engineered site), the engineered binding domain and binding partner (or "pair of binding partners") are useful in assays of enzymatic activity according to the invention.
Alternatively, candidate binding partners can be screened for their ability to bind the engineered binding domain in a modification-dependent manner. Such binding partners may be selected or designed based upon sequence homology with known binding partners or on molecular modeling data (e.g., from a modeling algorithm). Potential binding partners additionally may be purified (e.g, using the modified engineered binding domain as the trap in affinity chromatgraphy or as a probe for a library) from a population of polypeptide molecules. A library from which to draw a diverse population of polypeptide sequences of use in the invention includes, but is not limited to, an expression library or a synthetic peptide library (see "Candidate modulators", below).
One library-based technique which is useful in the invention to generate new pairs of assay components is that of phage display, which provides convenient testing of polypeptide sequences able to complex with the target sequence from a vast repertoire of different polypeptide sequences.
Filamentous bacteriophage display a small number of copies of a protein termed g3p on their surface. This protein is responsible for interacting with proteins on the surface of Escherichia coli and facilitates the infection of the bacterium. This protein possesses three globular domains linked by protease resistant, flexible amino acid sequences. The g3p protein can be modified to provide a means of presenting protein structures from which proteins capable of forming a stable binding complex can be identified. Such a bioassay can be configured in a number of ways including: a) Expression of the test proteins as an extension of the g3p sequence. Proteins able to bind with target polypeptide A can be selected by affinity purification on a matrix displaying polypeptide A. b) Expression of the test protein as an extension of the g3p protein, plus independent expression from the same phage of the target protein (polypeptide A) fused to a convenient affinity tag (such as His6). The binding of polypeptide A to the test protein displayed on g3p will facilitate the affinity purification of this phage particle. c) Expression of the test protein and polypeptide A as an interruption of the g3p sequence, preferably at one of the linker regions. This can produce fusion proteins of g3p N- term -polypeptide A and test protein partner B:g3p c-term, to which phage infective properties are only restored if polypeptide A and the test partner B bind to each other with reasonable affinity. This technique has been adapted from those previously described (Spada and Pluckthun, 1997, Nature Medicine. 3: 694-696; Sieber et al, 1998, Nature Biotechnology, 16: 955-960; Kristensen and Winter. 1998, Folding and Design, 3: 321-328).
If A is engineered to introduce a site for post-translational modification, then binding partners tolerant of that engineered site can be identified. A second round of selection can then be undertaken to identify the binding partners which dissociate upon post-translational modification of that site (i.e., those to which binding of the engineered binding domain is dependent upon post-translational modification).
Methods by which assays of the invention are performed using the engineered binding domains and binding partners therefor produced as described above are described in detail in the following sections and in the Example. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g, in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual. 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), chemical methods, pharmaceutical formulation and delivery and treatment of patients.
B. Methods to detect protei protein binding according to the invention
According to the invention, the activity of a modifying enzyme is assayed by measuring the formation or destruction of proteimprotein complexes when the modifying enzyme is present with an engineered binding domain, sequence or polypeptide and its corresponding binding partner under conditions which permit modifying activity. Methods which enable the detection of proteimprotein complexes (i.e., methods which allow one of skill in the art to discriminate between polypeptide pairing partners which are bound and those which are unbound) are known in the art. Of particular use in the invention are those methods which entail fluorescent labelling of the engineered domain, sequence or polypeptide and/or its binding partner, and subsequent detection of changes in fluorescence, whether in frequency or level, following incubation of the labeled assay components with the candidate modifying enzyme. Several such procedures are briefly summarized below.
Fluorescent resonance energy transfer (FRET)
A tool with which to assess the distance between one molecule and another (whether protein or nucleic acid) or between two positions on the same molecule is provided by the technique of fluorescent resonance energy transfer (FRET), which is now widely known in the art (for a review, see Matyus, 1992, J. Photochem. Photobiol. B: Biol. 12: 323-337, which is herein incorporated by reference). FRET is a radiationless process in which energy is transferred from an excited donor molecule to an acceptor molecule; the efficiency of this transfer is dependent upon the distance between the donor and acceptor molecules, as described below. Since the rate of energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, the energy transfer efficiency is extremely sensitive to distance changes. Energy transfer is said to occur with detectable efficiency in the 1-10 nm distance range, but is typically 4-6 nm for favorable pairs of donor and acceptor.
Radiationless energy transfer is based on the biophysical properties of fluorophores. These principles are reviewed elsewhere (Lakowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum Press, New York; Jovin and Jovin, 1989, Cell Structure and Function by Microspectrofluorometry, eds. E. Kohen and J.G. Hirschberg, Academic Press, both of which are incorporated herein by reference). Briefly, a fluorophore absorbs light energy at a characteristic wavelength. This wavelength is also known as the excitation wavelength. The energy absorbed by a flurochrome is subsequently released through various pathways, one being emission of photons to produce fluorescence. The wavelength of light being emitted is known as the emission wavelength and is an inherent characteristic of a particular fluorophore. Radiationless energy transfer is the quantum-mechanical process by which the energy of the excited state of one fluorophore is transferred without actual photon emission to a second fluorophore. That energy may then be subsequently released at the emission wavelength of the second fluorophore. The first fluorophore is generally termed the donor (D) and has an excited state of higher energy than that of the second fluorophore, termed the acceptor (A). The essential features of the process are that the emission specturm of the donor overlap with the excitation spectrum of the acceptor, and that the donor and acceptor be sufficiently close. The distance over which radiationless energy transfer is effective depends on many factors including the fluorescence quantum efficiency of the donor, the extinction coefficient of the acceptor, the degree of overlap of their respective spectra, the refractive index of the medium, and the relative orientation of the transition moments of the two fluorophores. In addition to having an optimum emission range overlapping the excitation wavelength of the other fluorophore, the distance between D and A must be sufficiently small to allow the radiationless transfer of energy between the fluorophores.
FRET may be performed either in vivo or in vitro. Proteins are labeled either in vivo or in vitro by methods known in the art. According to the invention, an engineered binding domain, sequence or polypeptide and its corresponding binding partner, comprised either by the same or by different polypeptide molecules, are differentially labeled, one with a donor and the other with an acceptor label, and differences in fluorescence between a test assay, comprising a protein modifying enzyme, and a control, in which the modifying enzyme is absent, are measured using a fluorimeter or laser-scanning microscope. It will be apparent to those skilled in the art that excitation/detection means can be augmented by the incorporation of photomultiplier means to enhance detection sensitivity. The differential labels may comprise either two different fluorescent labels (e.g., fluorescent proteins as described below or the fluorophores rhodamine, fluorescein, SPQ, and others as are known in the art) or a fluorescent label and a molecule known to quench its signal; differences in the proximity of the engineered binding domain, sequence or polypeptide and the binding partner with and without the protein-modifying enzyme can be gauged based upon a difference in the fluorescence spectrum or intensity observed.
This combination of protein-labelling methods and devices confers a distinct advantage over prior art methods for determining the activity of protein-modifying enzymes, as described above, in that results of all measurements are observed in real time (i.e., as a reaction progresses). This is significantly advantageous, as it allows both for rapid data collection and yields information regarding reaction kinetics under various conditions. A sample, whether in vitro or in vivo, assayed according to the invention therefore comprises a mixture at equilibrium comprising at least one* labeled engineered binding domain, sequence or polypeptide and its corresponding binding partner which, when disassociated from one another, fluoresce at one frequency and, when complexed together, fluoresce at another frequency or, alternatively, of molecules which either do or do not fluoresce depending upon whether or not they are associated.
A fluorescent label is either attached to the surface of the engineered binding domain, sequence or polypeptide or binding partner therefor or, alternatively, a fluorescent protein is fused in-frame with the engineered binding domain, sequence or polypeptide or binding partner therefor, as described below. The choice of fluorescent label will be such that upon excitation with light, labeled peptides which are associated will show optimal energy transfer between fluorophores. In the presence of a protein modifying enzyme (e.g., a phosphorylating-, a dephosphorylating-, a ubiquitinating-, ADP-ribosylating-, sentrinizing, prenylating- or glycosylating enzyme), a complex comprising an engineered binding domains, sequence or polypeptides and its binding partner dissociates due to structural or electrostatic disruption which occurs as a consequence of modification of the enzyme recognition site, thereby leading to a decrease in energy transfer and increased emission of light by the donor fluorophore. In this way, the state of polypeptide modification can be monitored and quantitated in real-time. This scheme, which represents the broadest embodiment of the invention, is shown in Figure 2.
As used herein, the terms "fluorophore" and "fluorochrome" refer interchangeably to a molecule which is capable of absorbing energy at a wavelength range and releasing energy at a wavelength range other than the absorbance range. The term "excitation wavelength" refers to the range of wavelengths at which a fluorophore absorbs energy. The term "emission wavelength" refers to the range of wavelength that the fluorophore releases energy or fluoresces.
A non-limiting list of chemical fluorophores of use in the invention, along with their excitation and emission wavelengths, is presented in Table 5.
Table 5
It should also be noted that where the fluorescent technique used requires both a donor molecule and an acceptor molecule, naturally occurring or engineered tryptophan residues within the binding domain and/or partner are suitable as a donor and therefore in this situation, only one of the molecules need be labelled with a fluorescent molecule.
Examples of fluorescent proteins which vary among themselves in excitation and emission maxima are listed in Table 1 of WO 97/28261 (Tsien et al., 1997, supra). These (each followed by [excitation max./emission max.] wavelengths expressed in nanometers) include wild-type Green Fluorescent Protein [395(475)/508] and the cloned mutant of Green Fluorescent Protein variants P4 [383/447], P4-3 [381/445], W7 [433(453)/475(501)], W2 [432(453)/480], S65T [489/511], P4-1 [504(396)/480], S65A [471/504], S65C [479/507], S65L [484/510], Y66F [360/442], Y66W [458/480], I0c [513/527], W1B [432(453)/476(503)], Emerald [487/508] and Sapphire [395/511]. This list is not exhaustive of fluorescent proteins known in the art; additional examples are found in the Genbank and SwissProt public databases. Further examples are described in Matz et al., 1999 (Nature Biotech 17: 969-973) and include red fluorescent protein from Discosoma sp. (drFP583). A number of parameters of fluorescence output are envisaged including
1) measuring fluorescence emitted at the emission wavelength of the acceptor (A) and donor (D) and determining the extent of energy transfer by the ratio of their emission amplitudes;
2) measuring the fluorescence lifetime of D;
3) measuring the rate of photobleaching of D;
4) measuring the anistropy of D and/or A; or
5) measuring the Stokes shift monomer; eximer fluorescence. Certain of these techniques are presented below.
Alternative fluorescent techniques suitable for monitoring proteimprotein binding in assays of the invention A one embodiment of the technology can utilize monomer:excimer fluorescence as the output. The association of an engineered binding domains, sequence or polypeptide with a binding partner in this format is shown in Figure 3.
The fluorophore pyrene when present as a single copy displays fluorescent emission of a particular wavelength significantly shorter than when two copies of pyrene form a planar dimer (excimer), as depicted. As above, excitation at a single wavelength (probably 340nm) is used to review the excimer fluorescence (~470nm) over monomer fluorescence (~375nm) to quantify assembly: disassembly of the reporter molecule.
Additional embodiments of the present invention are not dependent on FRET. For example the invention can make use of fluorescence correlation spectroscopy (FCS), which relies on the measurement of the rate of diffusion of a label (see Elson and Magde, 1974 Biopolymers, 13: 1-27; Rigler et al., 1992, in Fluorescence Spectroscopy: New Methods and Applications, Springer Verlag, pp.13-24; Eigen and Rigler, 1994, Proc. Natl. Acad. Sci. U.S.A., 91 : 5740-5747: Kinio and Rigler. 1995. Nucleic Acids Res.. 23: 1795-1799).
In FCS, a focused laser beam illuminates a very small volume of solution, of the order of 10"15 liter, which at any given point in time contains only one molecule of the many under analysis. The diffusion of single molecules through the illuminated volume, over time, results in bursts of fluorescent light as the labels of the molecules are excited by the laser.
Each individual burst, resulting from a single molecule, can be registered.
A labeled polypeptide will diffuse at a slower rate if it is large than if it is small. Thus, multimerized polypeptides will display slow diffusion rates, resulting in a lower number of fluorescent bursts in any given timeframe, while labeled polypeptides which are not multimerized or which have dissociated from a multimer will diffuse more rapidly. Binding of polypeptides according to the invention can be calculated directly from the diffusion rates through the illuminated volume. Where FCS is employed, rather than FRET, it is not necessary to label more than one polypeptide. Preferably, a single polypeptide member of the multimer is labeled. The labeled polypeptide dissociates from the multimer as a result of modification, thus altering the FCS reading for the fluorescent label.
A further detection technique which may be employed in the method of the present invention is the measurement of time-dependent decay of fluorescence anisotropy. This is described, for example, in Lacowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum Press, New York, incorporated herein by reference (see, for example, page 167).
Fluorescence anisotropy relies on the measurement of the rotation of fluorescent groups. Larger multimers of polypeptides rotate more slowly than monomers, allowing the formation of multimers to be monitored. The invention may be configured to exploit a number of non-fluorescent labels. In a first embodiment, the engineered binding domain and binding partner therefor form, when bound, an active enzyme which is capable of participating in an enzyme-substrate reaction which has a detectable endpoint. The enzyme may comprise two or more polypeptide chains or regions of a single chain, such that upon binding of the engineered binding domain to the binding partner, which are present either on two different polypeptide chains or in two different regions of a single polypeptide, these components assemble to form a functional enzyme. Enzyme function may be assessed by a number of methods, including scintillation counting and photospectroscopy. In a further embodiment, the invention may be configured such that the label is a redox enzyme, for example glucose oxidase, and the signal generated by the label is an electrical signal.
Modification of the engineered binding domain and, optionally, its binding partner according to the invention is required to inhibit binding and, consequently, enzyme component assembly, thus reducing enzyme activity.
In another assay format, an enzyme is used together with a modulator of enzyme activity, such as an inhibitor or a cofactor. In such an assay, one of the enzyme and the inhibitor or cofactor is an engineered binding domain, the other its binding partner. Binding of the enzyme to its inhibitor or cofactor results in modulation of enzymatic activity, which is detectable by conventional means (such as monitoring for the conversion of substrate to product for a given enzyme). Fluorescent protein labels in assays of the invention
In a FRET assay of the invention, the fluorescent protein labels are chosen such that the excitation spectrum of one of the labels (the acceptor) overlaps with the emission spectrum of the excited fluorescent label (the donor). The donor is excited by light of appropriate intensity within the donor's excitation spectrum. The donor then emits some of the absorbed energy as fluorescent light and dissipates some of the energy by FRET to the acceptor fluorescent label. The fluorescent energy it produces is quenched by the acceptor fluorescent label. FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor, reduction in the lifetime of its excited state, and re-emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor. When the donor and acceptor labels become spatially separated, FRET is diminished or eliminated.
One can take advantage of the FRET exhibited by an engineered binding domain, sequence or polypeptide and its corresponding binding partner labeled with different fluorescent protein labels, wherein one is linked to a donor and the other to an acceptor label, in monitoring protein modification according to the present invention. A single polypeptide may comprises a blue fluorescent protein donor label and a green fluorescent protein acceptor label, wherein one is fused to an engineered binding domain, sequence or polypeptide and the other is fused to its corresponding binding partner within that polypeptide; such a construct is herein referred to as a "tandem" fusion protein. Alternatively, two distinct polypeptides, ("single" fusion proteins) one comprising an engineered binding domain, sequence or polypeptide and the other its corresponding binding partner, may be differentially labeled with the donor and acceptor fluorescent protein labels, respectively. The construction and use of tandem fusion proteins in the invention can reduce significantly the molar concentration of peptides necessary to effect an association between differentially-labeled engineered species and their respective binding partners relative to that required when single fusion proteins are instead used. The labeled engineered binding domains, sequences or polypeptides and their corresponding binding partners may be produced via the expression of recombinant nucleic acid molecules comprising an in-frame fusion of sequences encoding an engineered binding domain, sequence or polypeptide or a binding partner therefor and a fluorescent protein label either in vitro (e.g., using a cell-free transcription/translation system, as described below, or instead using cultured cells transformed or transfected using methods well known in the art) or in vivo, for example in a trangenic animal including, but not limited to, insects, amphibians and mammals. A recombinant nucleic acid molecule of use in the invention may be constructed and expressed by molecular methods well known in the art, and may additionally comprise sequences including, but not limited to, those which encode a tag (e.g., a histidine tag) to enable easy purification, a secretion signal, a nuclear localization signal or other primary sequence signal capable of targeting the construct to a particular cellular location, if it is so desired.
The means by which the association between an engineered binding domain, sequence or polypeptide and its binding partner is assayed using fluorescent protein labels according to the invention may be briefly summarized as follows:
Whether or not the engineered binding domain, sequence or polypeptide and its binding partner are present on a single polypeptide molecule, one is labeled with a green fluorescent protein, while the other is preferably labeled with a red or, alternatively, a blue fluorescent protein. Useful donor: acceptor pairs of flurescent proteins (see Tsien et al., 1997, supra) include, but are not limited to:
Donor: S72A, K79R, Y145F, M153A and T203I (excitation λ 395nm; emission λ 511) Acceptor: S65G, S72A, K79R and T203Y (excitation λ 514 nm; emission λ 527), or T203Y/S65G, V68L, Q69K or S72A (excitation λ 515nm; emission λ 527nm).
An example of a blue:green pairing is P4-3 (shown in Table 1 of Tsien et al., 1997, supra) as the donor label and S65C (also of Table 1 of Tsien et al., 1997, supra) as the acceptor label. The mixtures comprising engineered binding domains, sequences or polypeptides and their corresponding binding partners are exposed to light at, for example, 368 nm, a wavelength that is near the excitation maximum of P4-3. This wavelength excites S65C only minimally. Upon excitation, some portion of the energy absorbed by the blue fluorescent protein label is transferred to the acceptor label through FRET if the engineered binding domain, sequence or polypeptide and its corresponding binding partner are in close association. As a result of this quenching, the blue fluorescent light emitted by the blue fluorescent protein is less bright than would be expected if the blue fluorescent protein existed in isolation. The acceptor label (S65C) may re-emit the energy at longer wavelength, in this case, green fluorescent light. After modification (e.g., phosphorylation, ADP-ribosylation, ubiquitination, prenylation, sentrination or glycosylation, all as described below) of one or both of the engineered binding domain, sequence or polypeptide and its binding partner by an enzyme, the two (and, hence, the green and red or, less preferably, green and blue fluorescent proteins) physically separate or associate, accordingly inhibiting or promoting FRET. For example, if activity of the modifying enzyme results in dissociation of a proteimprotein dimer, the intensity of visible blue fluorescent light emitted by the blue fluorescent protein increases, while the intensity of visible green light emitted by the green fluorescent protein as a result of FRET, decreases. Such a system is useful to monitor the activity of enzymes that modify the engineered binding domain, sequence or polypeptide or binding partner to which the fluorescent protein labels are fused as well as the activity of modulators or candidate modulators of those enzymes.
In particular, this invention contemplates assays in which the amount- or activity of a modifying enzyme in a sample is determined by contacting the sample with an engineered binding domain, sequence or polypeptide and its binding partner, differentially labeled with fluorescent proteins, as described above, and measuring changes in fluorescence of the donor label, the acceptor label or the relative fluorescence of both. Fusion proteins, as described above, which comprise either one or both labeled polypeptides comprising engineered binding domains, sequences or polypeptides and the corresponding binding partner of an assay of the invention can be used for, among other things, monitoring the activity of a modifying enzyme inside the cell that expresses the recombinant tandem construct or two different recombinant constructs.
Advantages of single- and tandem fluorescent fusion constructs include the greater extinction coefficient and quantum yield of many of these proteins compared with those of the Edans fluorophore. The acceptor in such a construct or pair of constructs is, itself, a fluorophore rather than a non-fluorescent quencher like Dabcyl.
Alternatively, in single-label assays of the invention, whether involving use of a chemical fluorophore or a single fluorescent fusion construct, such a non-fluorescent quencher may be used. Thus, the enzyme's substrate, i.e., the engineered binding domain, sequence or polypeptide (and, optionally, the binding partner) comprising a post-translational modification site and product (i.e., the engineered binding domain, sequence or polypeptide and its binding partner after addition or removal of a chemical moiety to/from the modification site) are both fluorescent, but with different fluorescent characteristics.
In particular, the substrate and modified products exhibit different ratios between the amount of light emitted by the donor and acceptor labels. Therefore, the ratio between the two fluorescences measures the degree of conversion of substrate to products, independent of the absolute amount of either, the thickness or optical density of the sample, the brightness of the excitation lamp, the sensitivity of the detector, etc. Furthermore, Aequorea-deήved or - related fluorescent protein labels tend to be protease resistant. Therefore, they are likely to retain their fluorescent properties throughout the course of an experiment.
Protein fusion constructs according to the invention
As stated above, recombinant nucleic acid constructs of particular use in the invention are those which comprise in-frame fusions of sequences encoding an engineered binding domain, sequence or polypeptide, and/or a binding partner therefor, and a fluorescent protein. If an engineered binding domain, sequence or polypeptide and its binding partner are to be expressed as part of a single polypeptide, the nucleic acid molecule additionally encodes, at a minimum, a donor fluorescent protein label fused to one, an acceptor fluorescent protein label fused to the other, a linker that couples them and is of sufficient length and flexibility to allow for folding of the polypeptide and pairing of the engineered binding domain, sequence or polypeptide and its binding partner and gene regulatory sequences operatively linked to the fusion coding sequence. If single fusion proteins are instead encoded (whether by one or more nucleic acid molecules), each nucleic acid molecule need only encode a polypeptide comprising an engineered domain, sequence or polypeptide or its binding partner fused either to a donor or acceptor fluorescent protein label and operatively linked to gene regulatory sequences.
"Operatively-linked" refers to polynucleotide sequences which are necessary to effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
As described above, a donor fluorescent protein label is capable of absorbing a photon and transferring energy to another fluorescent label. The acceptor fluorescent protein label is capable of absorbing energy and emitting a photon. Alternatively, a fluorophore emits fluorescent light which is absorbed by a quencher. If needed, the linker connects the engineered binding domain, sequence or polypeptide either directly or indirectly, through an intermediary linkage with one or both of the donor and acceptor fluorescent protein labels or the fluorescent label and, optionally, the quencher if a non-FRET assay is being performed. Regardless of the relative order of the engineered binding domain, sequence or polyepeptide or its binding partner and the donor and acceptor fluorescent protein labels on a polypeptide molecule, it is essential that sufficient distance be placed between the donor and acceptor or the fluorescent label and corresponding quencher by the linker and/or the engineered binding domain, sequence or polypeptide and corresponding binding partner to ensure that FRET does not occur unless the engineered binding domain, seqeunce or polypeptide and its binding partner dimerize. It is desirable, as described in greater detail in WO97/28261, to select a donor fluorescent protein label with an emission spectrum that overlaps with the excitation spectrum of an acceptor fluorescent protein label. In some embodiments of the invention the overlap in emission and excitation spectra will facilitate FRET. A fluorescent protein of use in the invention includes, in addition to those with intrinsic fluorescent properties, proteins that fluoresce due intramolecular rearrangements or the addition of cofactors that promote fluorescence.
For example, green fluorescent proteins ("GFPs") of cnidarians, which act as their energy-transfer acceptors in bioluminescence, can be used in the invention. A green fluorescent protein, as used herein, is a protein that fluoresces green light, and a blue fluorescent protein is a protein that fluoresces blue light. GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, from the sea pansy, Renilla reniformis, and from Phialidium gregarium. (Ward et al, 1982, Photochem. PhotobioL, 35: 803-808; Levine et al. 1982. Comp. Biochem. Phvsiol..72B: 77-85). A variety of Aequorea-r elated GFPs having useful excitation and emission spectra have been engineered by modifying the amino acid sequence of a naturally-occurring GFP from Aequorea victoria. (Prasher et al, 1992, Gene, 111 : 229-233; Heim et al., 1994, Proc. Natl. Acad. Sci. U.S.A.. 91: 12501-12504; PCTVUS95/ 14692). As used herein, a fluorescent protein is an Aequorea-related fluorescent protein if any contiguous sequence of 150 amino acids of the fluorescent protein has at least 85% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild-type Aequorea green fluorescent protein (SwissProt Accession No. P42212). Similarly, the fluorescent protein may be related to Renilla or Phialidium wild-type fluorescent proteins using the same standards.
Aequorea-related fluorescent proteins include, for example, wild-type (native) Aequorea victoria GFP, whose nucleotide and deduced amino acid sequences are presented in Genbank Accession Nos. L29345, M62654, M62653 and others Aequorea-related engineered versions of Green Fluorescent Protein, of which some are listed above. Several of these, i.e., P4, P4-3, W7 and W2 fluoresce at a distinctly shorter wavelength than wild type.
Recombinant nucleic acid molecules encoding single- or tandem fluorescent protein/polypeptide comprising engineered binding domain, sequences or polypeptides or their binding partners useful in the invention may be expressed either for in vivo assay of the activity of a modifying enzyme on the encoded products. Alternatively, the encoded fusion protiens may be isolated prior to assay, and instead assayed in a cell-free in vitro assay system, as described elsewhere herein.
C. Methods for detection of protein modification in real time i. In vitro protein modification and detection thereof Modifying enzymes The invention requires the presence of a modifying enzyme which catalyzes either the addition or removal of a modifying group. A range of kinases, phosphatases and other modifying enzymes are available commercially (e.g. from Sigma, St. Louis, MO; Promega, Madison, WI; Boehringer Mannheim Biochemicals, Indianapolis, IN; New England Biolabs, Beverly, MA; and others). Alternatively, such enzymes may be prepared in the laboratory by methods well known in the art. The catalytic sub-unit of protein kinase A (c-PKA) can be purified from natural sources (e.g. bovine heart) or from cells/organisms engineered to heterologously express the enzyme. Other isoforms of this enzyme may be obtained by these procedures. Purification is performed as previously described from bovine heart (Peters et al.,1977, Biochemistry, 16: 5691-5697) or from a heterologous source (Tsien et al., WO92/00388), and is in each case briefly summarized as follows:
Bovine ventricular cardiac muscle (2kg) is homogenized and then centrifuged. The supernatant is applied to a strong anion exchange resin (e.g. Q resin, Bio-Rad) equilibrated in a buffer containing 50mM Tris-HCl, lOmM NaCl, 4mM EDTA pH 7.6 and 0.2mM 2- mercaptoethanol. The protein is eluted from the resin in a second buffer containing 50mM Tris-HCl, 4mM EDTA pH 7.6, 0.2mM 2-mercaptoethanol, 0.5M NaCl. Fractions containing c-PKA are pooled and ammonium sulphate added to 30% saturation. Proteins precipitated by this are removed by centrifiigation and the ammonium sulphate concentration of the supernatant was increased to 75% saturation. Insoluble proteins are collected by centrifugation (included c-PKA) and are dissolved in 30mM phosphate buffer pH 7.0, lmM EDTA, 0.2mM 2-mercaptoethanol. These proteins are then dialysed against the same buffer (500 volume excess) at 4°C for two periods of 8 hours each. The pH of the sample is reduced to 6.1 by addition of phosphoric acid, and the sample is mixed sequentially with 5 batches of CM-Sepharose (Pharmacia, ~80 ml resin each) equilibrated in 30mM phosphate pH 6.1, lmM EDTA, 0.2 mM 2-mercaptoethanol. Cyclic AMP (10 μM) is added to the material which fails to bind to the CM-Sepharose, and the sample-cAMP mix is incubated with a fresh resin of CM-Sepharose (-100 ml) equilibrated as before. c-PKA is eluted from this column following extensive washing in equilibration buffer by addition of 30mM phosphate pH 6.1 , 1 mM EDTA, 1M KC1, 0.2 mM 2-mercaptoethanol. Fractions containing c-PKA are pooled and concentrated by filtration through a PM-30 membrane (or similar). The c-PKA sample is then subjected to gel-filtration chromatography on a resin such as Sephacryl 200HR (Pharmacia). The purification of recombinant c-PKA is as described in WO 92/00388. General methods of preparing pure and partially-purified recombinant proteins, as well as crude cellular extracts comprising such proteins, are well known in the art. Molecular methods useful in the production of recombinant proteins, whether such proteins are the enzymes to be assayed according to the invention or the labeled reporter engineered binding domains, sequences or polypeptides of the invention or their corresonding binding partners, are well known in the art (for methods of cloning, expression of cloned genes and protein purification, see Sambrook et al, 1989, supra; Ausubel et al., 1987-94, supra). The sequences of the catalytic subunit of several PKA molecules are found in the Genbank database (see PKA Cα, bovine, Genbank Accession Nos. X67154 and S49260; PKA Cβl, bovine, Genbank Accession No. J02647; PKA Cβ2, bovine, M60482, the form most likely purified from bovine heart by the protocol described above). According to the invention, assays of the activity of protein-modifying enzymes may be performed using crude cellular extracts, whether to test the activity of a recombinant protein or one which is found in nature, such as in a biological sample obtained from a test cell line or animal or from a clinical patient. In the former case, use of a crude cell extract enables rapid screening of many samples, which potentially finds special application in high- throughput screening methods, e.g. of candidate modulators of protein-modifying enzyme activity. In the latter case, use of a crude extract with the labeled reporter polypeptide comprising an engineered binding domain, sequence or polypeptide of the invention and a binding partner therefor facilitates easy and rapid assessment of the activity of an enzyme of interest in a diagnostic procedure, e.g., one which is directed at determining whether a protein-modifying enzyme is active at an a physiologically-appropriate level, or in a procedure designed to assess the efficacy of a therapy aimed at modulating the activity of a particular enzyme.
Production of polypeptides of use in the invention Engineered polypeptides, polypeptides comprising an engineered binding domain or sequence or binding partners for such engineered species may be synthesized by Fmoc or Tboc chemistry according to methods known in the art (e.g., see Atherton et al., 1981, J. Chem. Soc. Perkin I, 1981(2): 538-546; Merrifield, 1963, J. Am. Chem. Soc, 85: 2149- 2154, respectively). Following deprotection and cleavage from the resin, peptides are desalted by gel filtration chromatography and analyzed by mass spectroscopy, HPLC, Edman degradation and/or other methods as are known in the art for protein sequencing using standard methodologies.
Alternatively, nucleic acid sequences encoding such peptides may be expressed either in cells or in an in vitro transcription/translation system (see below) and, as with enzymes to be assayed according to the invention, the proteins purified by methods well known in the art.
Of particular use in the invention is phage display, in which an engineered binding domain is expressed from a phage chromosome along with on of a library of candidate binding partners. If a candidate binding partner binds the engineered binding domain, both are incorporated into the phage capsid.
Labelling polypeptides with fluorophores Engineered binding polypeptides, polypeptides comprising engineered binding domains or sequences, or binding partners therefor are labeled with thiol reactive derivatives of fluorescein and tetramethylrhodamine (isothiocyanate or iodoacetamide derivatives, Molecular Probes, Eugene, OR, USA) using procedures described by Hermanson G.T., 1995, Bioconjugate Techniques, Academic Press, London. Alternatively, primary-amine-directed conjugation reactions can be used to label lysine sidechains or the free peptide N-terminus (Hermason, 1995, supra).
Purification of fluorescent peptides
Fluorescent peptides are separated from unreacted fluorophores by gel filtration chromatography or reverse phase HPLC.
Phosphorylation of engineered binding domains and binding partners in vitro Peptides (0.01-l.OμM) are phosphorylated by purified c-PKA in 50mM Histidine buffer pH 7.0, 5mM MgSO4, lmM EGTA, 0.1-1.0 μM c-PKA, and 0.2mM [3 P] γ-ATP (specific activity ~2Bq/pmol) at 30-37°C for periods of time ranging from 0 to 60 minutes. Where the chemistry of the peptide is appropriate (i.e. having a basic charge) the phosphopeptide is captured on a cation exchange filter paper (e.g. phosphocellulose P81 paper; Whatman), and reactants are removed by extensive washing in 1% phosphoric acid (see Casnellie, 1991, Methods Enzymol., 200: 115-120). Alternatively, phosphorylation of samples is terminated by the addition of SDS-sample buffer (Laemmli, 1970, Nature, 227: 680-685) and the samples analyzed by SDS-PAGE electrophoresis, autoradiography and scintillation counting of gel pieces.
Dephosphorylation of engineered binding domains and binding partners in vitro The dephosphorylation of peptides phosphorylated as above is studied by removal of
ATP (through the addition of lOmM glucose and 30 U/ml hexokinase; Sigma, St. Louis, MO) and addition of protein phosphatase-1 (Sigma). Dephosphorylation is followed at 30-37°C by quantitation of the remaining phosphopeptide component at various time points, determined as above.
Fluorescence measurements of protein modification in vitro in real time Donor and acceptor fluorophore-labeled engineered binding polypeptides or polypeptides comprising engineered binding domains or sequences and the corresponding binding partners for any such engineered molecules (molar equivalents of fluorophore-labeled polypeptide or molar excess of acceptor-labeled polypeptide) are first mixed (if the two are present on separate polypeptides). Samples are analyzed in a fluorimeter using excitation wavelengths relevant to the donor fluorescent label and emission wavelengths relevant to both the donor and acceptor labels. A ratio of emission from the acceptor over that from the donor following excitation at a single wavelength is used to determine the efficiency of fluorescence energy transfer between fluorophores, and hence their spatial proximity. Typically, measurements are performed at 0-37 °C as a function of time following the addition of the modifying enzyme (and, optionally, a modulator or candidate modulator of function for that enzyme, as described below) to the system in 50mM histidine pH 7.0, 120 mM KC1, 5mM MgSO4, 5mM NaF, 0.05mM EGTA and 0.2mM ATP. The assay may be performed at a higher temperature if that temperature is compatible with the enzyme(s) under study.
Alternative cell-free assay system of the invention
A cell-free assay system must permit dimerization of an engineered binding domain, sequence or polypeptide with its binding partner to occur in a modification-dependent manner. As indicated herein, such a system may comprise a low-ionic-strengfh buffer (e.g., physiological salt, such as simple saline or phosphate- and/or Tris-buffered saline or other as described above), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate. The components of an assay of post-translational modification of a polypeptide molecule according to the invention may be added into a buffer, medium or lysate or may have been expressed in cells from which a lysate is derived. Alternatively, a cell-free transcription- and/or translation system may be used to deliver one or more of these components to the assay system. Nucleic acids of use in cell-free expression systems according to the invention are as described for in vivo assays, below. An assay of the invention may be peformed in a standard in vitro transcription/translation system under conditions which permit expression of a recombinant or other gene. The TNT® T7 Quick Coupled Transcription Translation System (Cat. # LI 170; Promega) contains all reagents necessary for in vitro transcription translation except the DNA of interest and the detection label; as discussed below, engineered binding domains, sequences or polypeptides and/or their binding partners may be encoded by expression constructs in which their coding sequences are fused in-frame to those encoding fluorescent proteins. The TNT® Coupled Reticulocyte Lysate Systems (comprising a rabbit reticulocyte lysate) include: TNT® T3 Coupled Reticulocyte Lysate System (Cat. # L4950; Promega); TNT® T7 Coupled Reticulocyte Lysate System (Cat. # L4610; Promega); TNT® SP6 Coupled Reticulocyte Lysate System (Cat. # L4600; Promega); TNT® T7/SP6 Coupled Reticulocyte Lysate System (Cat. # L5020; Promega); TNT® T7/T3 Coupled Reticulocyte Lysate System (Cat. # L5010; Promega).
An assay involving a cell lysate or a whole cell (see below) may be performed in a cell lysate or whole cell preferably eukaryotic in nature (such as yeast, fungi, insect, e.g., Drosophila), mouse, or human). An assay in which a cell lysate is used is performed in a standard in vitro system under conditions which permit gene expression. A rabbit reticulocyte lysate alone is also available from Promega, either nuclease-treated (Cat. # L4960) or untreated (Cat. # L4151).
Candidate modulators of protein-modifying enzymes to be screened according to the invention
Whether in vitro or in an in vivo system (see below), the invention encompasses methods by which to screen compositions which may enhance, inhibit or not affect (e.g., in a cross-screening procedure in which the goal is to determine whether an agent intended for one purpose additionally affects general cellular functions, of which protein modification is an example) the activity of a protein-modifying enzyme.
Candidate modulator compounds from large libraries of synthetic or natural compounds can be screened. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI). Combinatorial libraries are -available and can be prepared. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily produceable by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, preferably less than about 750, more preferably less than about 350 daltons. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like. Candidate modulators which may be screened according to the methods of the invention include receptors, enzymes, ligands, regulatory factors, and structural proteins. Candidate modulators also include nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens. Candidate modulators additionally comprise proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such as ribozymes or antisense nucleic acids). Proteins or polypeptides which can be screened using the methods of the present invention include hormones, growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structural proteins, viral antigens, parasitic antigens, bacterial antigens and antibodies (see below).
Candidate modulators which may be screened according to the invention also include substances for which a test cell or organism might be deficient or that might be clinically effective in higher-than-normal concentration as well as those that are designed to eliminate the translation of unwanted proteins. Nucleic acids of use according to the invention not only may encode the candidate modulators described above, but may eliminate or encode products which eliminate deleterious proteins. Such nucleic acid sequences are antisense RNA and ribozymes, as well as DNA expression constructs that encode them. Note that antisense RNA molecules, ribozymes or genes encoding them may be administered to a test cell or organism by a method of nucleic acid delivery that is known in the art, as described below. Inactivating nucleic acid sequences may encode a ribozyme or antisense RNA specific for the a target mRNA. Ribozymes of the hammerhead class are the smallest known, and lend themselves both to in vitro production and delivery to cells (summarized by Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S; Usman et al.. 1996. Curr. Qpin. Struct. Biol, 6: 527-533).
As stated above, antibodies are of use in the invention as modulators (specifically, as inhibitors) of protein-modifying enzymes. Methods for the preparation of antibodies are well known in the art, and are briefly summarized as follows: Either recombinant proteins or those derived from natural sources can be used to generate antibodies using standard techniques, well known to those in the field. For example, the proteins are administered to challenge a mammal such as a monkey, goat, rabbit or mouse.
The resulting antibodies can be collected as polyclonal sera, or antibody-producing cells from the challenged animal can be immortalized (e.g. by fusion with an immortalizing fusion partner) to produce monoclonal antibodies. 1. Polyclonal antibodies.
The antigen protein may be conjugated to a conventional carrier in order to increases its immunogenicity, and an antiserum to the peptide-carrier conjugate is raised. Coupling of a peptide to a carrier protein and immunizations may be performed as described (Dymecki et al.. 1992, J. Biol. Chem.. 267: 4815-4823). The serum is titered against protein antigen by ELISA (below) or alternatively by dot or spot blotting (Boersma and Van Leeuwen, 1994, J. Neurosci. Methods. 51 : 317). At the same time, the antiserum may be used in tissue sections prepared as described below. The serum is shown to react strongly with the appropriate peptides by ELISA, for example, following the procedures of Green et al., 1982, Cell, 28: 477-487. 2. Monoclonal antibodies.
Techniques for preparing monoclonal antibodies are well known, and monoclonal antibodies may be prepared using a candidate antigen whose level is to be measured or which is to be either inactivated or affinity-purified, preferably bound to a carrier, as described by Arnheiter et al., Nature, 294, 278-280 (1981).
Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue is introduced. Nevertheless, monoclonal antibodies may be described as being "raised to" or "induced by" a protein. Monoclonal antibody-producing hybridomas (or polyclonal sera) can be screened for antibody binding to the target protein. By antibody, we include constructions using the binding (variable) region of such an antibody, and other antibody modifications. Thus, an antibody useful in the invention may comprise a whole antibody, an antibody fragment, a polyfunctional antibody aggregate, or in general a substance comprising one or more specific binding sites from an antibody. The antibody fragment may be a fragment such as an Fv, Fab or F(ab')2 fragment or a derivative thereof, such as a single chain Fv fragment. The antibody or antibody fragment may be non-recombinant, recombinant or humanized. The antibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth. In addition, an aggregate, polymer, derivative and conjugate of an immunoglobulin or a fragment thereof can be used where appropriate.
Determination of activity of candidate modulator of a protein-modifying enzyme A candidate modulator of the activity of a protein-modifying enzyme may be assayed according to the invention as described herein, is determined to be effective if its use results in a difference of about 10%> or greater relative to controls in which it is not present (see below) in FRET resulting from the association of labeled engineered binding domains, sequences or polypeptides and their corresponding binding partner(s) in the presence of a protein-modifying enzyme.
The level of activity of a candidate modulator may be quantified using any acceptable limits, for example, via the following formula: (IndeXcontrol - Indexsampie) Percent Modulation = x 100
(IndeXcontrol)
where IndeXcontrol is the quantitative result (e.g., amount of- or rate of change in fluorescence at a given frequency, rate of molecular rotation, FRET, rate of change in FRET or other index of modification, including, but not limited to, enzyme inhibition or activation) obtained in assays that lack the candidate modulator (in other words, untreated controls), and Indexsampie represents the result of the same measurement in assays containing the candidate modulator. As described below, control measurements are made with a differentially-labeled engineered binding domain, sequence or polypeptide and its binding partner only and with these molecules plus a protein-modifying enzyme which recognizes a site present on them.
Such a calculation is used in either in vitro or in vivo assays performed according to the invention.
D. In vivo assays of enzymatic activity according to the invention Reporter group protein modification in living cells
Differentially-labeled engineered binding domains, sequences or polypeptides of the invention and their binding partners are delivered (e.g., by micro injection) to cells, such as smooth muscle cells (DDTl) or ventricular cardiac myocytes as previously described (Riabowol et al., 1988, Cold Spring Harbor Symposia on Quantitative Biology. 53: 85-90). The ratio of emission from the labeled molecule(s) is measured as described above via a photomultiplier tube focused on a single cell. Activation of a kinase (e.g., PKA by the addition of dibutyryl cAMP or β-adrenergic agonists) is performed, subsequent inhibition is performed by removal of stimulus and by addition of a suitable antagonist (e.g., cAMP antagonist Rp-cAMPS). As described elsewhere herein, an ADP ribosylating enzyme may be stimulated with cholera toxin (G-protein recognition feature) or with brefeldin A.
Heterologous expression of peptides Engineered binding domains, sequences or polypeptides and their binding partners can be produced from the heterologous expression of DNA sequences which encode them or may be chemically synthesized. Biological expression can be in procaryotic or eukaryotic cells using a variety of plasmid vectors capable of instructing heterologous expression. Purification of these products is achieved by destruction of the cells (e.g. French Press) and chromatographic purification of the products. This latter procedure can be simplified by the inclusion of an affinity purification tag at one extreme of the peptide, separated from the peptide by a protease cleavage site if necessary.
The use of cells or whole organisms in assays of the invention
When performed using cells, the assays of the invention are broadly applicable to a host cell susceptible to transfection or transformation including, but not limited to, bacteria (both gram-positive and gram-negative), cultured- or explanted plant (including, but not limited to, tobacco, arabidopsis, carnation, rice and lentil cells or protoplasts), insect (e.g., cultured Drosophila or moth cell lines) or vertebrate cells (e.g., mammalian cells) and yeast.
Organisms are currently being developed for the expression of agents including DNA, RNA, proteins, non-proteinaceous compounds, and viruses. Such vector microorganisms include bacteria such as Clostridium (Parker et al., 1947, Proc. Soc. Exp. Biol. Med., 66: 461-465; Fox et al., 1996, Gene Therapy. 3: 173-178; Minton et al., 1995, FEMS Microbiol. Rev.. 17: 357-364), Salmonella (Pawelek et al., 1997, Cancer Res.. 57: 4537-4544; Saltzman et al., 1996, Cancer Biother. Radiopharm.. 11 : 145-153; Carrier et al, 1992, J, Immunol.. 148: 1176-1181; Su et al., 1992. Microbiol. Pathol.. 13: 465-476; Chabalgoity et al., 1996, Infect. Immunol.. 65: 2402-2412), Listeria (Schafer et al., 1992, J. Immunol.. 149: 53-59; Pan et al., 1995, Nature Med.. 1 : 471-477) and Shigella (Sizemore et al, 1995, Science, 270: 299-302), as well as yeast, mycobacteria, slime molds (members of the taxa Dictyosteliida - such as of the genera Polysphondylium and Dictystelium, e.g. Dictyostelium discoideum - and Myxomycetes - e.g. of the genera Physarum and Didymium) and members of the Domain Arachaea (including, but not limited to, archaebacteria), which have begun to be used in recombinant nucleic acid work, members of the phylum Protista, or other cell of the algae, fungi, or any cell of the animal or plant kingdoms.
Plant cells useful in expressing polypeptides of use in assays of the invention include, but are not limited to, tobacco (Nicotiana plumb aginifolia and Nicotiana tabacum), arabidopsis (Arabidopsis thaliana), Aspergillus niger, Brassica napus, Brassica nigra, Datura innoxia, Vicia narbonensis, Viciafaba, pea (Pisum sativum), cauliflower, carnation and lentil (Lens culinaris). Either whole plants, cells or protoplasts may be transfected with a nucleic acid of choice. Methods for plant cell transfection or stable transformation include inoculation with Agrobacterium tumefaciens cells carrying the construct of interest (see. among others, Turpen et al., 1993, J. Virol. Methods, 42: 227-239), administration of liposome-associated nucleic acid molecules (Maccarrone et al, 1992, Biochem. Biophvs. Res. Commun.. 186: 1417-1422) and microparticle injection (Johnston and Tang, 1993, Genet. Eng. (NY), 15: 225-236), among other methods. A generally useful plant transcriptional control element is the cauliflower mosaic virus (CaMV) 35S promoter (see, for example. Saalbach et al., 1994, Mol. Gen. Genet, 242: 226-236). Non-limiting examples of nucleic acid vectors useful in plants include pGSGLUCl (Saalbach et al., 1994, supra), pGA492 (Perez et al., 1989, Plant Mol. Biol. 13: 365-373), pOCA18 (Olszewski et al., 1988, Nucleic Acids Res.. 16: 10765-10782), the Ti plasmid (Roussell et al., 1988, Mol. Gen. Genet. 211 : 202-209) and pKR612Bl (Balazs et al, 1985, Gene, 40: 343-348).
Mammalian cells are of use in the invention. Such cells include, but are not limited to, neuronal cells (those of both primary explants and of established cell culture lines) cells of the immune system (such as T-cells, B-cells and macrophages), fibroblasts, hematopoietic cells and dendritic cells. Using established technologies, stem cells (e.g. hematopoietic stem cells) may be used for gene transfer after enrichment procedures. Alternatively, unseparated hematopoietic cells and stem cell populations may be made susceptible to DNA uptake. Transfection of hematopoietic stem cells is described in Mannion-Henderson et al., 1995, Exp. Hematol., 23: 1628; Schiffmann et al., 1995, Blood, 86: 1218; Williams, 1990, Bone Marrow Transplant. 5: 141; Boggs, 1990, Int. J. Cell Cloning, 8: 80; Martensson et al, 1987, Eur. J. Immunol.. 17: 1499; Okabe et al., 1992, Eur. J. Immunol.. 22: 37-43; and Banerji et al., 1983, Cell, 33: 729. Such methods may advantageously be used according to the present invention.
Nucleic acid vectors for the expression of assay components of the invention in cells or multicellular organisms
A nucleic acid of use according to the methods of the invention may be either double- or single stranded and either naked or associated with protein, carbohydrate, proteoglycan and/or lipid or other molecules. Such vectors may contain modified and/or unmodified nucleotides or ribonucleotides. In the event that the gene to be transfected may be without its native transcriptional regulatory sequences, the vector must provide such sequences to the gene, so that it can be expressed once inside the target cell. Such sequences may direct transcription in a tissue-specific manner, thereby limiting expression of the gene to its target cell population, even if it is taken up by other surrounding cells. Alternatively, such sequences may be general regulators of transcription, such as those that regulate housekeeping genes, which will allow for expression of the transfected gene in more than one cell type; this assumes that the majority of vector molecules will associate preferentially with the cells of the tissue into which they were injected, and that leakage of the vector into other cell types will not be significantly deleterious to the recipient mammal. It is also possible to design a vector that will express the gene of choice in the target cells at a specific time, by using an inducible promoter, which will not direct transcription unless a specific stimulus, such as heat shock, is applied.
A gene encoding a component of the assay system of the invention or a candidate modulator of protein-modifying enzyme activity may be transfected into a cell or organism using a viral or non- viral DNA or RNA vector, where non- viral vectors include, but are not limited to, plasmids, linear nucleic acid molecules, artificial chromomosomes and episomal vectors. Expression of heterologous genes in mammals has been observed after injection of plasmid DNA into muscle (Wolff J. A. et al, 1990, Science, 247: 1465-1468; Carson D.A. et al., US Patent No. 5,580,859), thyroid (Sykes et al., 1994, Human Gene Ther., 5: 837-844), melanoma (Vile et al, 1993, Cancer Res., 53: 962-967), skin (Hengge et al, 1995, Nature Genet., 10: 161-166), liver (Hickman et al., 1994, Human Gene Therapy, 5: 1477-1483) and after exposure of airway epithelium (Meyer et al., 1995, Gene Therapy, 2: 450-460).
In addition to vectors of the broad classes described above and gene expression constructs encoding fusion proteins comprising engineered binding domains, sequences or polypeptides fused in-frame with fluorescent proteins as described above (see "Fluorescent resonance energy transfer"), microbial plasmids, such as those of bacteria and yeast, are of use in the invention.
Bacterial plasmids:
Of the frequently used origins of replication, pBR322 is useful according to the invention, and pUC is preferred. Although not preferred, other plasmids which are useful according to the invention are those which require the presence of plasmid encoded proteins for replication, for example, those comprising pT 181, FII, and FI origins of replication. Examples of origins of replication which are useful in assays of the invention in E coli and S typhimurium include but are not limited to, pHETK (Garapin et al., 1981, Proc. Natl. Acad. Sci. U.S.A., 78: 815-819), p279 (Talmadge et al., 1980. Proc. Natl. Acad. Sci. U.S.A., 77: 3369-3373), p5-3 and p21A-2 (both from Pawalek et al., 1997, Cancer Res., 57: 4537-4544), pMBl (Bolivar et al., 1977, Gene, 2: 95-1 13), ColEl (Kahn et al., 1979, Methods Enzvmol.. 68: 268-280), pl5A (Chang et al, 1978, J. Bacteriol., 134: 1141-1 156); pSClOl (Stoker et al., 1982, Gene, 18: 335-341); R6K (Kahn et al., 1979, supra); Rl (temperature dependent origin of replication, Uhlin et al., 1983, Gene, 22: 255-265); lambda dv (Jackson et al., 1972, Proc. Nat Aca. Sci. U.S.A., 69: 2904-2909); pYA (Nakayama et al., 1988, infra). An example of an origin of replication that is useful in Staphylococcus is pT181 (Scott, 1984, Microbial Reviews 48: 1-23). Of the above-described origins of replication, pMBl, pl5A and ColEl are preferred because these origins do not require plasmid-encoded proteins for replication. Yeast plasmids: Three systems are used for recombinant plasmid expression and replication in yeasts:
1. Integrating. An example of such a plasmid is Yip, which is maintained at one copy per haploid genome, and is inherited in Mendelian fashion. Such a plasmid, containing a gene of interest, a bacterial origin of replication and a selectable gene (typically an antibiotic-resistance marker), is produced in bacteria. The purified vector is linearized within the selectable gene and used to transform competent yeast cells. Regardless of the type of plasmid used, yeast cells are typically transformed by chemical methods (e.g. as described by Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The cells are treated with lithium acetate to achieve transformation efficiencies of approximately 104 colony-forming units (transformed cells)/μg of DNA. Yeast perform homologous recombination such that the cut, selectable marker recombines with the mutated (usually a point mutation or a small deletion) host gene to restore function. Transformed cells are then isolated on selective media.
2. Low copy-number ARS-CEN, of which YCp is an example. Such a plasmid contains the autonomous replicating sequence (ARS1), a sequence of approximately 700 bp which, when carried on a plasmid, permits its replication in yeast, and a centromeric sequence (CEN4), the latter of which allows mitotic stability. These are usually present at 1-2 copies per cell. Removal of the CEN sequence yields a YRp plasmid, which is typically present in
100-200 copes per cell; however, this plasmid is both mitotically and meiotically unstable.
3. High-copy-number 2μ circles. These plasmids contain a sequence approximately
1 kb in length, the 2μ sequence, which acts as a yeast replicon giving rise to higher plasmid copy number; however, these plasmids are unstable and require selection for maintenance.
Copy number is increased by having on the plasmid a selection gene operatively linked to a crippled promoter. This is usually the LEU2 gene with a truncated promoter (LEU2-d), such that low levels of the Leu2p protein are produced; therefore, selection on a leucine- depleted medium forces an increase in copy number in order to make an amount of Leu2p sufficient for cell growth.
As suggested above, examples of yeast plasmids useful in the invention include the YRp plasmids (based on autonomously-replicating sequences, or ARS) and the YEp plasmids (based on the 2μ circle), of which examples are YEp24 and the YEplac series of plasmids (Gietz and Sugino, 1988, Gene. 74: 527-534). (See Sikorski, "Extrachromsomoal cloning vectors of Saccharomyces cerevisiae", in Plasmids. A Practical Approach. Ed. K.G. Hardy, IRL Press, 1993 ; and Yeast Cloning Vectors and Genes. Current Protocols in Molecular Biology. Section II, Unit 13.4, Eds., Ausubel et al, 1994).
In addition to a yeast origin of replication, yeast plasmid sequences typically comprise an antibiotic resistance gene, a bacterial origin of replication (for propagation in bacterial cells) and a yeast nutritional gene for maintenance in yeast cells. The nutritional gene (or "auxotrophic marker") is most often one of the following (with the gene product listed in parentheses and the sizes quoted encompassing the coding sequence, together with the promoter and terminator elements required for correct expression):
TRP1 (PhosphoADP-ribosylanthranilate isomerase, which is a component of the tryptophan biosynthetic pathway).
URA3 (Orotidine-5'-phosphate decarboxylase, which takes part in the uracil biosynthetic pathway).
LEU2 (3-Isopropylmalate dehydrogenase, which is involved with the leucine biosynthetic pathway). HIS3 (Imidazoleglycerolphosphate dehydratase, or IGP dehydratase).
LYS2 (α-aminoadipate-semialdehyde dehydrogenase, part of the lysine biosynthetic pathway). Alternatively, the screening system may operate in an intact, living multicellular organism, such as an insect or a mammal. Methods of generating transgenic Drosophila. mice and other organisms, both transiently and stably, are well known in the art; detection of fluorescence resulting from the expression of Green Fluorescent Protein in live Drosophila is well known in the art. One or more gene expression constructs encoding one or more of a labeled engineered binding domain, sequence or polypeptide, a binding partner therefor, a protein-modifiying enzyme and, optionally, a candidate modulator thereof are introduced into the test organism by methods well known in the art (see also below). Sufficient time is allowed to pass after administration of the nucleic acid molecule to allow for gene expression, for binding of engineered binding domains, sequences or polypeptides and their binding partners, and for chromophore maturation, if necessary (e.g., Green Fluorescent Protein matures over a period of approximately 2 hours prior to fluorescence) before FRET is measured. A reaction component (particularly a candidate modulator of enzyme function) which is not administered as a nucleic acid molecule may be delivered by a method selected from those described below.
Dosage and administration of a labeled engineered binding domain, sequence or polypeptide. a binding partner and protein-modifying enzyme or candidate modulator thereof for use in an in vivo assay of the invention i. Dosage
For example, the amount of each labeled engineered binding domain or binding partner therefor must fall within the detection limits of the fluorescence-measuring device employed. The amount of an enzmye or candidate modulator thereof will typically be in the range of about lμg - 100 mg kg body weight. Where the candidate modulator is a peptide or polypeptide, it is typically administered in the range of about 100 - 500 μg/ml per dose. A single dose of a candidate modulator, or multiple doses of such a substance, daily, weekly, or intermittently, is contemplated according to the invention.
A candidate modulator is tested in a concentration range that depends upon the molecular weight of the molecule and the type of assay. For example, for inhibition of protein/protein or protein/DNA complex formation or transcription initiation (depending upon the level at which the candidate modulator is thought or intended to modulate the activity of a protein modifying enzyme according to the invention), small molecules (as defined above) may be tested in a concentration range of lpg - 100 μg/ml, preferably at about 100 pg - 10 ng/ml; large molecules, e.g., peptides, may be tested in the range of 10 ng - 100 μg/ml, preferably 100 ng - 10 μg/ml.
Generally, nucleic acid molecules are administered in a manner compatible with the dosage formulation, and in such amount as will be effective. In the case of a recombinant nucleic acid encoding a engineered binding domain and/or binding partner therefor, such an amount should be sufficient to result in production of a detectable amount of the labeled protein or peptide, as discussed above. In the case of a protein modifying enzyme, the amount produced by expression of a nucleic acid molecule should be sufficient to ensure that at least 10% of engineered binding domains or binding partners therefor will undergo modification if they comprise a target site recognized by the enzyme being assayed. Lastly, the amount of a nucleic acid encoding a candidate modulator of a protein modifying enzyme of the invention must be sufficient to ensure production of an amount of the candidate modulator which can, if effective, produce a change of at least 10%> in the effect of the target protein modifying enzyme on FRET resulting from binding of a engineered binding domain to its binding partner or, if administered to a patient, an amount which is prophylactically and/or therapeutically effective.
When the end product (e.g. an antisense RNA molecule or ribozyme) is administered directly, the dosage to be administered is directly proportional to the amount needed per cell and the number of cells to be transfected, with a correction factor for the efficiency of uptake of the molecules. In cases in which a gene must be expressed from the nucleic acid molecules, the strength of the associated transcriptional regulatory sequences also must be considered in calculating the number of nucleic acid molecules er target cell that will result in adequate levels of the encoded product. Suitable dosage ranges are on the order of, where a gene expression construct is administered, 0.5- to lμg, or 1- lOμg, or optionally 10- 100 μg of nucleic acid in a single dose. It is conceivable that dosages of up to lmg may be advantageously used. Note that the number of molar equivalents per cell vary with the size of the construct, and that absolute amounts of DNA used should be adjusted accordingly to ensure adequate gene copy number when large constructs are injected. If no effect (e.g., of a modifying enzyme or an inhibitor thereof) is seen within four orders of magnitude in either direction of the starting dosage, it is likely that an enzyme does not recognize the target site of the engineered binding domain (and, optionally, its binding partner) according to the invention, or that the candidate modulator thereof is not of use according to the invention. It is critical to note that when high dosages are used, the concentration must be kept below harmful levels, which may be known if an enzyme or candidate modulator is a drug that is approved for clinical use. Such a dosage should be one (or, preferably, two or more) orders of magnitude below the LD50 value that is known for a laboratory mammal, and preferably below concentrations that are documented as producing serious, if non-lethal, side effects.
Components of screening assays of the invention may be formulated in a physiologically acceptable diluent such as water, phosphate buffered saline, or saline, and further may include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. Administration of labeled polypeptides comprising a engineered binding domain, sequence, polypeptide or a binding partner therefor, a protein kinase or phosphatase or a candidate modulator as described herein may be either localized or systemic.
Localized adminstration:
Localized administration of a component of an assay of the invention is preferably by via injection or by means of a drip device, drug pump or drug- saturated solid matrix from which the nucleic acid can diffuse implanted at the target site. When a tissue that is the target of delivery according to the invention is on a surface of an organism, topical administration of a pharmaceutical composition is possible.
Compositions comprising a composition of- or of use in the invention which are suitable for topical administration can take one of several physical forms, as summarized below: (i) A liquid, such as a tincture or lotion, which may be applied by pouring, dropping or "painting" (i. e. spreading manually or with a brush or other applicator such as a spatula) or injection.
(ii) An ointment or cream, which may be spread either manually or with a brush or other applicator (e.g. a spatula), or may be extruded through a nozzle or other small opening from a container such as a collapsible tube.
(iii) A dry powder, which may be shaken or sifted onto the target tissue or, alternatively, applied as a nebulized spray. (iv) A liquid-based aerosol, which may be dispensed from a container selected from the group that comprises pressure-driven spray bottles (such as are activated by squeezing), natural atomizers (or "pump-spray" bottles that work without a compressed propellant) or pressurized canisters. (v) A carbowax or glycerin preparation, such as a suppository, which may be used for rectal or vaginal administration of a therapeutic composition.
In a specialized instance, the tissue to which a candidate modulator of a protein kinase or phosphatase is to be delivered for assay (or, if found effective, for therapeutic use) is the lung. In such a case the route of administration is via inhalation, either of a liquid aerosol or of a nebulized powder of. Drug delivery by inhalation, whether for topical or systemic distribution, is well known in the art for the treatment of asthma, bronchitis and anaphylaxis.
In particular, it has been demonstrated that it is possible to deliver a protein via aerosol inhalation such that it retains its native activity in vivo (see Hubbard et al., 1989, J. Clin.
Invest, 84: 1349-1354).
Systemic administration:
Systemic administration of a protein, nucleic acid or other agent according to the invention may be performed by methods of whole-body drug delivery are well known in the art. These include, but are not limited to, intravenous drip or injection, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug-impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device, which may comprise a reservoir of exogenously-produced protein, nucleic acid or other material or may, instead, comprise cells that produce and secrete a engineered binding domain and/or a binding partner therefor, modifying enzyme or candidate modulator thereof. Note that injection may be performed either by conventional means (i.e. using a hypodermic needle) or by hypospray (see Clarke and Woodland, 1975, Rheumatol. RehabiL. 14: 47-49). Components of assays of the invention can be given in a single- or multiple dose.
Delivery of a nucleic acid may be performed using a delivery technique selected from the group that includes, but is not limited to, the use of viral vectors and non-viral vectors, such as episomal vectors, artificial chromosomes, liposomes, cationic peptides, tissue-specific cell transfection and transplantation, administration of genes in general vectors with tissue- specific promoters, etc.
E. Kits according to the invention i. A kit for assaying the activity of a protein-modifying enzyme
In order to facilitate convenient and widespread use of the invention, a kit is provided which contains the essential components for screening the activity of a an enzyme which mediates a change in protein modification, as described above. A labeled, engineered binding domain, sequence or polypeptide, as defined above, and a differentially labeled binding partner which binds it specifically in a modification-dependent manner is provided, as is a suitable reaction buffer for in vitro assay or, alternatively, cells or a cell lysate. A reaction buffer which is "suitable" is one which is permissive of the activity of the enzyme to be assayed and which permits modification dependent binding of the engineered binding domain, sequence or polypeptide and the binding partner. The labeled components are provided as peptide/protein or a nucleic acid comprising a gene expression construct encoding the one or more of a peptide/protein, as discussed above. Polypeptides in a kit of the invention are supplied either in solution (preferably refrigerated or frozen) in a buffer which inhibits degradation and maintains biological activity, or are provided in dried form, i.e., lyophilized. In the latter case, the components are resuspended prior to use in the reaction buffer or other biocompatible solution (e.g. water, containing one or more of physiological salts, a weak buffer, such as phophate or Tris, and a stabilizing substance such as glycerol, sucrose or polyethylene glycol); in the latter case, the resuspension buffer should not inhibit modification-dependent protein binding when added to the reaction buffer in an amount necessary to deliver sufficient protein for an assay reaction. Polypeptides provided as nucleic acids are supplied- or resuspended in a buffer which permits either transfection/transformation into a cell or organism or in vitro transcription/translation, as described above. Each of these components is supplied separately contained or in admixture with one or more of the others in a container selected from the group that includes, but is not limited to, a tube, vial, syringe or bottle. Optionally, the kit includes cells. Eukaryotic or prokaryotic cells, as described above, are supplied in- or on a liquid or solid physiological buffer or culture medium (e.g. in suspension, in a stab culture or on a culture plate, e.g. a Petri dish). For ease of shipping, the cells are typically refrigerated, frozen or lyophilized in a bottle, tube or vial. Methods of cell preservation are widely known in the art; suitable buffers and* media are widely known in the art, and are obtained from commerical suppliers (e.g., Gibco/LifeTechnologies) or made by standard methods (see, for example Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
An enzyme being assayed according to the invention is added to the assay system either as a protein (isolated, partially-purified or present in a crude preparation such as a cell extract or even a living cell) or a recombinant nucleic acid. Methods of expressing a nucleic acid comprising an enzyme or other protein are well known in the art (see again above). An assay of the invention is carried out using the kit according to the methods described above, in the Example and elsewhere.
ii. A kit for screening a candidate modulator of protein-modifying enzyme activity A candidate modulator of post-translational modification may be assayed using a kit of the invention. A kit as described above is used for this application, with the assay performed further comprising the addition of a candidate modulator of the modifying enzyme which is present to the reaction system. Optionally, a protein-modifying enzyme is supplied with the kit, either as a protein or nucleic acid as described above.
Assays of protein activity are performed as described above. At a minimum, three detections are performed, one in which the engineered binding domain, sequence or polypeptide and its binding partner are present without the modifying enzyme or candidate modulator thereof (control reaction A), one in which the same polypeptide components are incubated with the modifying enzyme under conditions which permit the modification reaction to occur (control reaction B) and one in which the modifying enzyme and candidate inhibitor are both incubated with the labeled engineered binding domain, sequence or polypeptide and corresponding binding partner under conditions which permit the modification reaction to occur (test reaction). The result of the last detection procedure is compared with those of the first two controls; the candidate inhibitor is judged to be efficacious if there is a shift observed amount or rate of change in total fluorescence, FRET, mass of a protein complex or inhibition or activation of an enzyme of at least 10%> away from that observed in control reaction B toward that observed in control reaction A. Example 1 - Generation of an engineered binding domain/binding partner pair for use in a protein binding assay of enzymatic activity (cAMP dependent protein kinase) according to the invention. As stated above, a test protein may be expressed as an extension of the g3p protein, while a target protein (polypeptide A) for the test protein is fused to a convenient affinity tag (such as His6) and expressed in the same phage. The binding of polypeptide A to the test protein displayed on g3p facilitates the affinity purification of the phage particle. This example illustrates reduction of the invention to practice using the strategy described in option 2. The binding partner structures can be derived from heterodimeric protein complexes, homodimeric protein complexes or from two domains of a single polypeptide which have been cleaved into separate entities for the purpose of this invention.
The protein backbone of ubiquitin (Ub), a 76-residue cellular protein of known tertiary structure, can be broken at one of a number of locations without significant loss of structure. In this cleaved state, the two halves of the ubiquitin protein remain together in a compact and stable complex (Johnsson and Varshavsky, 1994, Proc. Natl. Acad. Sci U.S.A., 91 : 10340-10344). In the following discussion, these N- and C- terminal fragments of the ubiquitin protein are be referred to as "sUb-N" (for the N-terminal fragment of the split ubiquitin) and "sUb-C" (for the C-terminal fragment of split ubiquitin). A site for post-translational modification is introduced into sUb-N which could disrupt complex formation when that site is modified, and mutagenesis of sUb-C is then performed to identify a variant of it which is capable of binding the engineered sUb-N.
Mammalian Ub has been cloned into a phage-display vector system, pCANTABb, developed at the University of Sussex from the Pcanbab5 VECTOR. Ub can be expressed from this system and displayed on the surface of filamentous phage (Finucane et al (1999) Biochemistry 36 1 1604-11612 Finucane & Woolfson (1999) Biochem 36 11613-11623). This vector system has been used to prepare constructs that direct the expression of the sUb fragments and, in turn, these are used to generate the required mutants.
Specifically, the split has been positioned at Asp39, which is a highly exposed residue in the native Ub structure. Residues 20-23 of sUb-N, which have the sequence SDTI are be mutated to RRKS to be recognised and phosphorylated to PKA. The SD to RR mutations should not destabilize the sUb complex significantly, because in the native structure, these residues are highly exposed to solvent. The 123 to S mutation is expected to be destabilizing, because 123 is a hydrophobic residue located in the core of the native protein. Thus, in addition to the chain break, this mutation may destabilize the sUb sufficiently to prevent complexation of sUb-N and sUb-C. To accommodate the polar Ser mutation and generate a stable sUb complex, residues Leu43, Leu50 and Leu56 of sUb-C, which are also in the core of the protein and contact 123 in native ubiquitin, are mutated to all combinations of the 20 amino acids to give a library of 8,000 mutations.
The overall sequence changes are as follows:
Ub
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQSKEGIPPDQQRLIFAGKQLEDGRTLSDY
NIQKESTLHLVLRLRGG
sUb-N
MQIFVKTLTGKTITLEVEARRKSENNKAKJQDKEGIPP
sUb-C DQQRXIFAGKQXEDGRTXSDYΝIQKESTLHLVLRLRGG
Stable sUb complexes are selected from this pool as follows: sUb-Ν containing the RRKS mutation are expressed with an Ν-terminal hexahistidine tag to allow it to be captured onto surfaces with chelated Νi, such as a BIAcore chip used in surface plasmon resonance or Νi-agarose beads. The mutant sUb-Cs are made as a fusion with g3p to allow them to be displayed on surface of filamentous phage. Thus, when the protein- phage library is passed over the sequestered mutant sUb-Ν, particles that interact strongly with this fragment are be captured, but those that show no affinity for sUb-Ν are be washed away. The separated His6-sUb-Ν:sUb-C-phage complexes could then be broken using protein denaturants such as urea or guanidinium chloride that do not disrupt the phage. This are permit elution of the phage, which are then be amplified by re-infection into E-coli. This panning and amplification procedure is repeated a number of times in order to amplify mutant sUb-Cs that bind tightly to the sUb-N mutant and rescue sUb complexation. The selected mutants are identified by sequencing of the selected phage DNA.
The split Ub fragment His6-sUb-N and sUb-C-phage have been obtained. The former binds effectively to BIAcore chips derivatised with Ni. Moreoever, passing the sUb-C-phage over the bound His6-sUb-N gave binding consistent with 1 : 1 complex formation (see Figure
4). In addition, we demonstrated that the resulting complex is less stable than an intact
(unsplit) Ub-phage fusion in a proteolysis experiment.
Construction of phagemid vector systems The DNA coding the complete ubiquitin sequence was amplified from a rat DNA library using the PCR, Taq polymerase (Boeringer Mannheim) and a standard protocol. The primers (5'-GTACTGCAGGGAATGCAGATCTTCGTGAAGACC-3' and
S'-AATCTCGAGACCGCCGCCCCTCAGGCGGAGGAC 3') are used to add 5' (Pstl) and 3' (Xhol) restriction sites and terminal glycine codons. The PCR product was ligated into the pGEM-T vector (Promega) and sub-cloned into an in-house vector, pCANTABb, using the Pstl and Xhol sites. pCANTABb was derived from the phage-display vector pCANTAB5 by adding the coding sequence for a hexahistidine tag 5' to the g3p sequence (McCafferty et al, 1994, Appl. Biochem. Biotech., 47: 157-173).
The pCANTABb-Ub construct directed the expression of the protein fusion: g3p periplasmic leader sequence-hexahistidine tag- LQG-Ub section 1 ; and the independent production of gill periplasmic leader sequence-Ub-GLDQQ-g3p. The construct for sUb-N was prepared from pCANTABb-Ub by eliminating the C-terminal half of the protein Asp39-Gly76, using Kunkel mutagenesis (Kunkel, 1985, Proc. Natl. Acad. Sci. U.S.A., 82: 488-492) and the mutagenic primer 5'-TGCGGCCGCCTACTAAGGGGGGATGCCCTC-3'. The construct for sUb-C was prepared from pCANTABb-Ub by sticky-feet mutagenesis (Clackson and Winter, 1989, Nucleic Acids Res., 17(24): 10163-10170) using the primer 5-GAGCCTCTGCTGGTCGGCCATGGCCGGCTG-3', which effectively excises the DNA coding sUb-N. Mutagenesis of Ub-N to insert PKA phosphorylation site
The codons for residues 20-23 (sequence SDTI) of wild-type sUb-N were mutated in PCANTABb-sUb-N using Kunkel mutagenesis (Kunkel, 1985, supra) and the primer: 5'-CTTCACGTTCTCGCTCTTGCGGCGGGGCTCCACCTC-3'.
Construction of the phagemid library
The library of sUb-C is prepared from PCANTABb-sUb-C by mutating the codons for Leu43, Leu50 and Leu56 to NN[GC], where N = 1 : 1 : 1 : 1 mix for G:A:C:T. This codes for all 20 amino acids in the following proportions: F, W, Y, C, I, M, K, N, H, Q, D, E, 3L, 3R, 3S, 2V, 2 A, 2G, 2P, 2T + 1 amber stop codon.
Kunkel mutagenesis (Kunkel, 1985, supra) is used with the following mutagenic primer:
5'-GTAATCAGA[GC]NNGGTGCGGCCATCTTC[GC]NNCTGCTTGCCGGCAAAGA T[GC]NNCCTCAGCAT-3'
Phage display, biopanning and protease-selection
Phage for biopanning and analysis are prepared as follows: XL 1 -blue E-coli cells, transformed with appropriate vectors, are superinfected with Ml 3 helper phage and grown overnight at 37 C. Phage are precipitated from the growth supernatant using 20%>PEG/2.5M NaCl, resuspended in 1000 ml of buffer A (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and clarified briefly by centrifugation. For the selection studies on the phage library, four rounds of panning are typically performed. In general, conditions for panning are established using surface plasmon resonance in BIAcore. Ni-derivatized NTA chips are used for this purpose along with standard running buffers and protocols. After the chips are derivatized with Ni, purified His6-sUb-N carrying the PKA recognition site is added and bound to the chip surface. Phage displaying the library of sUb-C mutants are passed over the fully derivatized chip and binding (sUb complex formation) is monitored by changes in the SPR signal. This method is also useful in preparative phage selection; however, for displayed libraries with more mutants, preparative panning is done on Ni-agarose beads. A typical protocol for this is: 100 ml of phage-library preparations are mixed with 800 ml of buffer and 250 ml of Ni-NTA agarose beads. Phage are allowed to bind to the beads for 10 minutes at room temperature. Beads are then washed a number of times (e.g. at least 5) with 750 ml of buffer, and the supernatant removed by centrifiigation at each stage. Phage bound to the beads are then eluted with buffer containing 250 mM imidazole, and amplified and purified as described above. The sequences of the selected sUb-C sequences that bind the mutant sUb-N peptide are determined from amplified phage by isolating and sequencing the phagemid DNA.
Identification of Ub complexes upset by PKA phosphorylation Phage expressing sUb-C that bind the sUb-N mutant and thus form sUb complexes tolerant of the PKA phosphorylation site (in the dephosphorylated form) are loaded onto Ni agarose beads as described above, washed and resuspended in 50mM HEPES pH 7.0, 120mM KCI, 5mM MgSO4, lmM ATP and lmM PKA (catalytic subunit), and the mixture is incubated at 372C for a period of 5 minutes. In this procedure, the majority of sUb complexes do not tolerate the addition of phosphate and, thus, phage are released from the beads into the supernatant, which is recovered by centrifiigation. Phage are amplified and purified as described above for sequencing. In this way, sequences for sUb that tolerate the PKA site in its non-phosphorylated form, but not in its phosphorylated form, are determined.
Use of binding partners identified by phage display in an assay of the invention The component polypeptides of a 'binding pair' comprising an engineered binding domain and its binding partner, such as those identified through phage display as above, are expressed and purified by molecular and biochemical known to one of ordinary skill in the art. At least one of the engineered binding domain and the binding partner is labelled with a detectable label, as described above. The engineered binding domain is contacted with the binding partner in a buffer or other medium which permits modification-dependent proteimprotein binding (binding that occurs specifically when the site for post-translational modification is in one modification state but not the other). Methods by which to assess proteimprotein binding (e.g., FRET, fluorescence correlation spectroscopy, monomer: excimer fluorescence, fluorescence anisotropy, determination of mass or monitoring of enzymatic activity) are performed, both in the presence and absence of modifying enzyme, candidate modifying enzyme (i.e., an enzyme of unknown function) or a biological sample whose enzymatic activity is assayed according to the methods of the invention. According to one useful technique, the engineered binding domain, which is not labelled with a detectable label, is immobilized and then contacted with the binding partner, where the partner is still attached to a phage particle from the phage display procedure. Interactions between the engineered domain and the binding partner are monitored through the partner protein still attached to the phage particle (surface plasmon resonance). Alternatively, FCS is used if the pure protein component is fluorescent, rather than immobilized. In the case of Ub-N and Ub-C, interactions are monitored with Ub-C expressed on the surface of phage. A difference of at least 10%> in surface plasmon resonance of fluorescence emission respectively observed in the presence of the modifying enzyme, candidate modifying enzyme or biological sample relative to that observed in its absence indicates that the enzyme or sample being tested has protein-modifying activity.
Example 2 - PKA assay using engineered ZAP70 SH2 domain and binding partner derived from T-cell receptor zeta chain The assay described herein is based on the concept that the tandem SH2 domain from
ZAP binds the dually tyrosine phosphorylated motif from the zeta chain of the T-cell receptor (TCRζ). Binding only occurs when the TCRζ is phosphorylated on both tyrosine residues, i.e. the interaction between one phospho-tyrosine and one of the tandem SH2 domains is not enough to give stringent binding. Immobilised assays have been performed to demonstrate binding of a TCR peptide to the tandem SH2 domain of ZAP. A protein kinase A (PKA) site is introduced into the ZAP protein, in close proximity to one of the phospho-tyrosine binding pockets, to enable phosphorylation by PKA. Upon phosphorylation of the engineered protein by PKA, the presence of the large negatively charged phosphate group on ZAP prevents the interaction between the binding pocket and the phospho-tyrosine residue, therefore blocking the ZAP-TCR interaction.
Vector construction
Primers are designed based on the published ZAP-70 DNA sequence (Genbank accession number L05148). The SH2 domain (amino acids 1-259) of ZAP70 was cloned by PCR using the following oligo-nucleotides:
Forward Primer 1 : GGGATCCATATGCCAGACCCCGCGGCGCACCTG Reverse Primer 1 : GGAATTCGGGCACTGCTGTTGGGGCAGGCCTCC
The resultant PCR fragment is digested with Ndel and EcoRl and inserted into pET28a (Novagen) to generate vector pFS45. DNA encoding GFP in the vector pQBI25-FNI (Quantum) is digested with M and the resultant, single stranded 5' overhang is "filled in" using T4 DNA polymerase (NEB) to generated complete, double stranded DNA. After the polymerase is denatured by heat treatment the DNA is further digested with EcoRl and the resultant 850 bp band was gel purified. The vector pFS45 is digested with Hindlll and the resultant 5' overhang is "filled in" with T4 DNA polymerase and then further digested with EcoRl. After the digested vector is gel purified it is ligated with the purified DNA encoding GFP to generate pFS46, which is designed to express a ZAP70-GFP fusion protein in bacteria.
Expression and purification procedure Fresh transformants of pFS46 in BRL(DE3) pLysS are used to inoculate 3ml
LB/kanamycin (lOOμg/ml). The starter cultures are incubated overnight at 37°C with shaking. From these starter cultures 1ml is used to inoculate 400ml Terrific Brofh/kanamycin (lOOμg/ml) in a 2L, baffled flask. Cultures are incubated at 37°C at 200 rpm for approximately 5 hrs until the OD 600nm had reached 0.5 Abs units. At this point cultures are induced by adding IPTG to a concentration of lmM. The cultures are then left incubating at room temperature overnight with gentle shaking on a benchtop rotator.
Bacteria are harvested by centrifugation at 3000 rpm for 20 min. The bacterial pellet is resuspended in 25ml lysis buffer (50mM Pi pH 7.0, 300mM NaCl, 2% Proteinase inhibitor cocktail (Sigma), 0.75mg ml Lysozyme). Lysis of the resuspended cells is initiated by gentle stirring for 1 hr. at room temperature. The partially lysed mixture is subjected to 2 cycles of freeze thawing in liquid nitrogen. Finally the cells are sonicated on ice using a 10mm probe at high power. Sonication is performed on a pulse setting for a period of 3 min. The crude lysate is then centrifuged at 15000 rpm for 30 min. to remove cell debris. His tagged proteins are purified from the clear lysate using TALON® resin (Clontech). Proteins are bound to the resin in a batchwise manner by gentle shaking at room temperature for 30 min. Non-His tagged proteins are removed by washing the resin at least twice with lOx bed volume of wash buffer (50mM sodium phosphate pH 7.0, 300mM NaCl, 5mM fluorescence-blank Imidazole). The washed resin was loaded into a 2 ml column and the bound proteins released with elution buffer (50mM sodium phosphate pH 7.0, 300mM NaCl, 150mM florescence-blank Imidazole). Elution is normally achieved after the first 0.5ml and within 2-3ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10% glycerol.
Immobilized assay
Biotinylated peptides are immobilised in a 96-well streptavidin coated plate
(Advanced Biotechnologies) using 1.0 mg/ml peptide in TBS containing 0.2%> Tween 20 and 1% BSA (TBST). For immobilization, peptide (200ml) is incubated in the wells for lhr at room temperature with gentle agitation on a rotating platform. Excess peptide is removed with 3 washes of 200 ml of TBST.
Phosphorylated TCRζ chain peptide: RCKFSRSAEPPAYQQGQNQLY(p)NELNLGRREEY(p)DVLD
Unphosphorylated TCRζ chain peptide: RCKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLD
The presence of phosphorylated peptide is detected by adding 200ml of ZAPGFP (lmM) TBST/BSA and incubating for 1 hour at RT with gentle rocking. This is followed by 6 washes of 200ml TBST. Bound ZAPGFP is detected by reading the fluorescence of GFP using excitation at 485nm and measuring emission at 520nm. (Figure 5).
Engineered binding domain assay using an immobilised assay with a natural binding partner labelled with GFP.
ZAPPKA-GFP Vector construction: To introduce a PKA site within ZAP in close proximity to one of the tyrosine binding pockets, residues 207-210 (TVYH) are mutated to RRAS. The following oligos are used in the mutagenesis:
Forward primer 2: CGCGCTAGCTACCTCATCAGCC
Reverse primer 2 : GGGGCTAGCGCGCCGCTTCCC ATAGATGAGGG These oligos used in tandem with the original cloning oligos create two ZAP fragments which can be joined together by the Nhel site (indicated in bold) present in the primer 2 set. The ZAPPKA fragment is then inserted into pFS46 to generate a vector which is designed to express a ZAPPKA-GFP fusion protein in bacteria.
Protein purification
Proteins are purified as described above for ZAP-GFP.
Immobilized assay The immobilized assay is performed as described above. It is first necessary to demonstrate that ZAPPKA-GFP binds in an equivalent manner to ZAP-GFP. Once this is confirmed, ZAP-GFP and ZAPPKA-GFP are pre-phosphorylated using 1.5pmol of PKA in 200 ml PKA buffer (50mM Histidine buffer pH 7.0, 5mM MgSO4, 120mM KC1, 5mM NaF, lmM ATP, 0.2mg/ml BSA) for 1 hr at 30°C. After phosphorylation, the immobilized assays are performed as before. Pre-phosphorylation is expected to have no effect on ZAP-GFP binding but is contemplated to completely inhibit binding of ZAPPKA-GFP to the immobilized TCRζ peptide. The presence of the phosphorylated peptide is detected by the fluorescence of GFP as described above.
It is contemplated according to the invention that binding will be detected for the ZAP-GFP and the non-phosphorylated ZAPPKA-GFP, while the PKA-phosphorylated ZAPPKA-GFP will display little to no binding.
Engineered binding domain assay using an immobilised assay with a natural binding partner labelled with a coiled-coil tag and a fluorescent detector molecule (ZAP70-FJ-fluorescein peptide).
Vector construction
The ZAP70SH2 domain is constructed as a fusion protein with a coiled-coil peptide based on the Fos/Jun coiled-coil peptide. Oligonucleotides based on the coiled-coil domain of Fos/Jun have been designed and synthesised: Forward primer:
GGGGGGAGCTCTGGGAGGCGGAGGTGGAGGGCTGATGCGCCAGCTGCAGGATG
AAGTTGAAGAACTGGAACAGGAAAACTGGCATCTGCAGA
Reverse primer:
CCCCCCTCGAGTTATTAAACTTCGGCTTCCAGGCACTGAACTTCACGCAGCAGAC GGGCAACTTCGTTCTGCAGATGCCAGTTTTCCTGTTCCAGT
This DNA encodes a glycine linker followed by the coiled-coil polypeptide sequence (Polypeptide FJ):
LMRQLQDEVEELEQENWHLQNEVARLLREVQCLEAEV
The primers are annealed together by heating to 96°C followed by slow cooling to room temperature. Complete double stranded DNA is generated by "filling in" the single stranded 5' overhangs using Klenow fragment of DNA polymerase I (NEB). The DNA fragment is purified by electrophoresis in 1.2% agarose gel and DNA is extracted from an isolated gel band using Qiagen spin columns. The purified fragment is digested with Sad and Xhol and purified as above prior to ligation into the bacterial expression vector pET28a to generate vector FS101. FS101 is digested with Nhel and EcoRI and DNA encoding ZAPPKA is ligated into this plasmid to generate a vector that is designed to express ZAPPKA-Fos/Jun.
ZAPPKA-FJ purification and labelling with fluorescein
The ZAPPKA-FJ purification and labelling with fluorescein is according to the methods described above for ZAP GFP except that the IPTG induction is at room temperature for 90 mins.
The corresponding synthetic Fos/Jun polypeptide partner that forms the coiled-coil pair with ZAPPKA-Fos/Jun is prepared:
RMRQLEDRVEELREQNWHLANQVARLRQRVCELKARV Peptide domains can be specifically labeled on amine or thiol groups with chemical fluorophores such as fluorescein or rhodamine. Fluorophores with thiol or amine reactive chemistries are readily available from commercial sources such as Molecular Probes. These fluorophores can be conjugated to peptides under mild conditions (e.g. 20mM TES pH 7 for thiol directed labeling, or 200mM sodium bicarbonate pH 8.3 for amine directed labeling, using 230μM peptide in the presence of 200μM label).
The peptide is labeled with fluorescein through amine directed labeling. Purified ZAPPKA-FJ is mixed with the peptide and the mixture morritored by FP to detect coiled-coil formation between the F and J peptides, resulting in ZAPPKA labeled with fluorescein. The immobilized assay is then performed according to the procedure described above.
It is first necessary to demonstrate that ZAPPKA-FJ binds in an equivalent manner to
ZAP-GFP. Once this is confirmed, ZAP-GFP and ZAPPKA-FJ are pre-phosphorylated using
1.5pmol of PKA in 200 ml PKA buffer (50mM Histidine buffer pH 7.0, 5mM MgS04,
120mM KC1, 5mM NaF, lmM ATP, 0.2mg/ml BSA) for 1 hr at 30°C. After phosphorylation the immobilized assays are be performed as before. Pre-phosphorylation is expected to have no effect on ZAP-GFP binding but completely inhibit binding of ZAPPKA-FJ to the immobilized TCRζ peptide.
It is contemplated according to the invention that binding will be detected for the ZAP-GFP and the non-phosphorylated ZAPPKA-FJ, while the PKA-phosphorylated ZAPPKA-FJ will display little to no binding.
USE
The invention is useful in monitoring the activity of a protein-modifying enzyme. whether the protein is isolated, partially-purified, present in a crude preparation or present in a living cell. The invention is further useful in assaying a cell or cell extract for the presence- or level of activity of a protein modifying enzyme. The invention is additionally useful in assaying the activity of naturally-occurring (mutant) or non-natural (engineered) isoforms of known protein modifying enzymes or, instead, that of novel (natural or non-natural) enzymes. The invention is of use in assaying the efficacy of candidate modulators of the activity of a protein modifying enzyme in inhibiting or enhancing the activity of that enzyme; moreover, is useful to screen potential therapeutic drugs for activity against cloned and/or purified enzymes that may have important clinical pathogenicities when mutated. The invention is further of use in the screening of candidate bioactive agents (e.g., drugs) for side effects, whereby the ability of such an agent to modulate the activity of a protein modifying enzyme may be indicative a propensity toward provoking unintended side-effects to a therapeutic or other regimen in which that agent might be employed.
OTHER EMBODIMENTS
Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.

Claims

1. An isolated engineered binding domain and a binding partner therefor, wherein said isolated engineered binding domain includes a site for post-translational modification and binds a binding partner therefor in a manner dependent upon modification of said site.
2. The isolated engineered binding domain and binding partner therefor of claim 1, wherein said site comprises a sequence which directs modification by one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase, a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase and an NAD:Arginine ADP ribosyltransferase.
3. The isolated engineered binding domain and binding partner therefor of claim 2, wherein said site permits addition of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and said addition prevents binding of said isolated engineered binding domain to said binding partner.
4. The isolated engineered binding domain and binding partner therefor of claim 2, wherein said site permits addition of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and said addition promotes binding of said isolated engineered binding domain to said binding partner.
5. The isolated engineered binding domain and binding partner therefor of claim 2, wherein said site permits removal of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and said removal prevents binding of said isolated engineered binding domain to said binding partner.
6. The isolated engineered binding domain and binding partner therefor of claim 2, wherein said site permits removal of a chemical moiety which may be: a phosphate moiety, a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acyl moiety and a sentrin moiety, and said removal promotes binding of said isolated engineered binding domain to said binding partner.
7. The isolated engineered binding domain and binding partner therefor of claim 1, wherein at least one of said isolated engineered binding domain and said binding partner comprises a detectable label.
8. The isolated engineered binding domain and binding partner therefor of claim 7, wherein said detectable label emits light.
9. The isolated engineered binding domain and binding partner therefor of claim 8, wherein said light is fluorescent.
10. The isolated engineered binding domain and binding partner therefor of claim 9, wherein one of said isolated engineered binding domain and said binding partner comprises a quencher for said detectable label.
1 1. A kit comprising an isolated engineered binding domain and a binding partner therefor, wherein said isolated engineered binding domain includes a site for post- translational modification and binds said binding partner in a manner dependent upon modification of said site, and packaging materials therefor.
12. The kit of claim 11, wherein said kit further comprises a buffer which permits modification-dependent binding of said isolated engineered binding domain and said binding partner.
13. The kit of claim 12, wherein said buffer additionally permits modification of said site for protein modification by one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase, a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase, and an NAD:Arginine ADP ribosyltransferase.
14. The kit of claim 1 1, wherein said kit further comprises one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase, a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase, and an NAD:Arginine ADP ribosyltransferase.
15. The kit of claim 14, wherein said kit further comprises a substrate for said enzyme which may be: MgATP, ubiquitin, sentrin, nicotinamide adenine dinucleotide (NAD4), uridine-diphosphate-N-acetylglucosamine-dolichyl-phosphate (UDP-N-acetylglucosamine- dolichyl-phosphate), palmytyl CoA, myristoyl CoA and UDP-N-acetylglucosamine.
16. The kit of claim 14, wherein said kit further comprises a cofactor for said enzyme.
17. The kit of claim 11 , wherein at least one of said isolated engineered binding domain and said binding partner comprises a detectable label.
18. The kit of claim 17, wherein said detectable label emits light.
19. The kit of claim 18, wherein said light is fluorescent.
20. A method for monitoring activity of an enzyme comprising performing a detection step to detect binding of an isolated engineered binding domain and a binding partner therefor as a result of contacting one or both of said isolated engineered binding domain and said binding partner with said enzyme, wherein said isolated engineered binding domain includes a site for post-translational modification and binds said binding partner in a manner dependent upon modification of said site and wherein detection of binding of said isolated engineered binding domain and said binding partner as a result of said contacting is indicative of enzyme activity.
21. A method for monitoring activity of an enzyme comprising performing a detection step to detect dissociation of an isolated engineered binding domain from a binding partner therefor as a result of contacting one or both of said isolated engineered binding domain and said binding partner with said enzyme, wherein said isolated engineered binding domain includes a site for post-translational modification and binds said binding partner in a manner dependent upon modification of said site and wherein detection of dissociation of said isolated engineered binding domain from said binding partner as a result of said contacting is indicative of enzyme activity.
22. The method of claim 20 or 21 , wherein at least one of said isolated engineered binding domain and said binding partner is labeled with a detectable label.
23. The method of claim 22, wherein said label emits light.
24. The method of claim 23, wherein said light is fluorescent.
25. The method of claim 22, wherein said detection step is to detect a change in signal emission by said detectable label.
26. The method according to claim 25, wherein said method further comprises exciting said detectable label and monitoring fluorescence emission.
27. The method according to claim 20 or 21, wherein said enzyme is one of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase, a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase and an NAD:Arginine ADP ribosyltransferase.
28. The method according to claim 20 or 21 , wherein said method further comprises the step, prior to or after said detection step, of contacting said isolated engineered binding domain and said binding partner with an agent which modulates the activity of said enzyme.
29. A method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase, a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase and an NAD:Arginine ADP ribosyltransferase, the method comprising
(a) contacting an isolated engineered binding domain, a binding partner therefor and an enzyme with a candidate modulator of said enzyme, wherein said engineered binding domain includes a site for post-translational modification and binds said binding partner in a manner that is dependent upon modification of said site by said enzyme, and wherein at least one of said isolated engineered binding domain and said binding partner comprises a detectable label, and
(b) monitoring the binding of said isolated engineered binding domain to said binding partner, wherein binding or dissociation of said isolated engineered binding domain and said binding partner as a result of said contacting is indicative of modulation of enzyme activity by said candidate modulator of said enzyme.
30. The method according to claim 29, wherein said detectable label emits light.
31. The method according to claim 30, wherein said light is fluorescent.
32. The method according to claim 31, wherein said monitoring comprises measuring a change in energy transfer between a label present on said isolated engineered binding domain and a label present on said binding partner.
33. A method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a kinase, a phosphatase, a carbohydrate transferase, a ubiquitin activating enzyme El, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase and an NAD:Arginine ADP ribosyltransferase, the method comprising
(a) contacting an assay system with a candidate modulator of enzymatic activity of a said enzyme, and
(b) monitoring binding of an isolated engineered binding domain and a binding partner therefor in said assay system, wherein said engineered binding domain includes a site for post-translational modification and binds said binding partner in a manner that is dependent upon modification of said site by at least one said enzyme in said assay system, wherein at least one of said isolated engineered binding domain and said binding partner comprises a detectable label, and wherein binding or dissociation of said isolated engineered binding domain and said binding partner as a result of said contacting is indicative of modulation of enzyme activity by said candidate modulator of a said enzyme.
34. The method according claim 20, 21 , 29 or 33, wherein said method comprises realtime observation of association of a said isolated engineered binding domain and its binding partner.
EP00906478A 1999-02-25 2000-02-25 Compositions and methods for monitoring the modification of engineered binding partners Withdrawn EP1155321A1 (en)

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