Phage Display
Phage Display
Phage Display
Marjorie Russel
The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
1 Introduction
Display of peptides and proteins on lamentous phagephage displayis an in vitro selection technique that enables polypeptides with desired properties to be extracted from a large collection of variants. A gene of interest is fused to that of a phage coat protein, resulting in phage particles that display the encoded protein and contain its gene, providing a direct link between phenotype and genotype. This allows phage libraries to be subjected to a selection step (e.g. afnity chromatography), and recovered clones to be identied by sequencing and re-grown for further rounds of selection. Since the initial description of the approach by Smith (1), it has become established as a powerful method for identifying polypeptides with novel properties, and altering the properties of existing ones (for reviews, see 25). Filamentous phages are ideal in many ways for use as cloning vehicles and for display in particular. The genome is small and tolerates insertions into nonessential regions; cloning and library construction are facilitated by the ability to isolate both single- and double-stranded DNA (ssDNA and dsDNA), and by the availability of simple plasmid-based vectors; coat proteins can be modied with retention of infectivity; phage can accumulate to high titers since their production does not kill cells; and phage particles are stable to a broad range of potential selection conditions. This chapter is intended to provide the background needed to initiate a phage display project. In the rst portion (Sections 2 and 3), the life cycle, genetics, and 1
MARJORIE RUSSEL ET AL. structural biology of lamentous phages are summarized, with a focus on aspects that are relevant to phage display. We then go on to describe general considerations to be made when approaching a new phage display project, including choice of display format, experimental design, and common pitfalls (Sections 46). Finally, summaries of commercial sources of phage display vectors, kits, and alternative display systems are provided for those cases in which such reagents can provide a head-start for investigators (Sections 7 and 8). Cross-references are provided to later chapters in the book that provide detailed procedures.
Ff bacteriophage E.coli
PS
Figure 1 Life cycle of lamentous phage f1 (M13/fd). Sequential binding of pIII to the tip of the F-pilus and then the host Tol protein complex results in depolymerization of the phage coat proteins, their deposition in the cytoplasmic membrane (where they are available for reutilization), and entry of the ssDNA into the cytoplasm. The ssDNA is converted by host enzymes to a double-stranded RF, the template for phage gene expression. Progeny ssDNA, coated by pV dimers (except for the packaging sequence hairpin (PS) that protrudes from one end), is the precursor of the virion. A multimeric complex that spans both membranes composed of pI, pXI, pIV, and the cytoplasmic host protein thioredoxinmediates conversion of the pVssDNA complex to virions and secretion of virions from the cell. This process involves removal of pV dimers and their replacement by the ve coat proteins that transiently reside in the cytoplasmic membrane.
the C-terminus of pVIII are at the inner surface of the tube and interact with phosphates of the viral ssDNA. The ends of the particle are distinguishable in electron micrographs. The blunt end contains several (35) copies each of pVII and pIX, two of the smallest ribosomally translated proteins known (33 and 32 residues, respectively). Neither their structure nor disposition in the particle is known. However, immunological evidence indicates that at least some of pIX is exposed (11) and antibody variable regions have been successfully displayed on the amino termini of pVII and pIX (12). Phage assembly begins at the pVIIpIX end, and in the absence of either protein, no particle is formed. The pointed end of the particle contains about ve copies each of pIII and pVI, both of which are needed in order for the phage to detach from the cell membrane; pVI is degraded in cells that lack pIII, which suggests that these proteins 3
II/X
III
VI
I/IX
IV
Replication
Assembly/export
Figure 2 Filamentous phage f1 (M13/fd) genes and gene products. Gene II encodes pII, which binds in the IG region (located between genes IV and II/X; not shown) of dsDNA and makes a nick in the strand, initiating replication by host proteins. pX is required later in infection for the switch to ssDNA accumulation. Gene V encodes the ssDNA binding protein pV. Genes VII and IX encode two small proteins located at the tip of the virus that is rst to emerge from the cell during assembly. Gene VIII encodes the major coat protein, and genes III and VI encode pIII and pVI, which are located at the end of the virion and mediate termination of assembly, release of the virion, and infection. Gene I encodes two required cytoplasmic membrane proteins, pI and pXI, and gene IV encodes pIV, a multimeric outer membrane channel through which the phage exits the bacterium. Note that the genome is in fact circular, but is shown in a linear presentation here for clarity.
assemble in the cell membrane before their incorporation into phage particles (13). They can be isolated from phage as a complex (14). The disposition of pVI in the particle is not known, but pVI with fusions to the C-terminus can be incorporated into phage, suggesting that this portion of the 112-residue pVI may be surface exposed (13). More is known about the 406-residue pIII, the most commonly used coat protein for display (Figure 3). Its N-terminal domain, which is necessary for phage infectivity, is surface exposed and forms the small ``knobs'' that can often be seen to emanate from the pointed end of the particle in electron micrographs. Three pIII domains have been dened, the two N-terminal of which (N1 and N2) are believed to interact intramolecularly, based on crystallographic analysis (16, 17). The three domains are separated by two long, presumably exible linkers characterized by repeats of a glycine-rich sequence. The nal 132 residues within the C-terminal CT domain are necessary and sufcient for pIII to be incorporated into the phage particle and to mediate termination of assembly and release of phage from the cell; this domain is likely to be buried within the particle (13). The single-stranded phage genome is oriented within the phage particle. Its orientation is determined by the packaging signal (PS), located in the non-coding IG region of the genome. The PS, an imperfect but extremely stable hairpin, is positioned at the pVIIpIX end of the particle and is necessary and sufcient for efcient encapsidation of circular ssDNA into phage particles. Certain amino acid 4
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY substitutions in pVII, pIX, and pI (see below) enable single strands that lack a PS to be encapsidated; it is not known whether the DNA is randomly oriented in such particles or if some small duplex region serves as a secondary PS.
2.3 Infection
All lamentous phages that have been characterized use pili, which are long and slender cell surface appendages that resemble the phage themselves, as receptors. There are many different kinds of pili. E. coli phage use self-transmissible pili that mediate transfer of the plasmid that encodes them to recipient bacteria. Ff phage bind F pili, and IKe uses N and P pili. Phage can infect cells that lack appropriate pili, but the process is extremely inefcient; the efciency is improved two to four orders of magnitude by agents that concentrate the phage or promote its adherence to the cell surface, such as CaCl2 and polyethylene glycol (18). Infection normally begins when the N2 domain of pIII (Figure 3) binds to the tip of a pilus (19) (see Figure 4). As might be expected from the low abundance of F pili (only a few per cell) and the small target size that their ends present, the rate and efciency of infection of a bacterial culture is improved at high multiplicity of phage per cell and high cell density. However, if high density is achieved by growing cells past log phase (as opposed to concentrating them gently), pilus expression is decreased and infectivity compromised. Furthermore, since F pili do not assemble at low temperatures, efcient infection (and therefore plaque formation) by Ff requires incubation at or above 34 C. Pili normally assemble and disassemble continuously, and this, possibly stimulated by phage binding, brings the phage close to the cell surface. Upon pilus binding to N2, the N1 domain is released from its normal interaction with N2, making it available to bind to the host TolA protein, which extends into the periplasm from the cytoplasmic membrane (20, 21). Thus, the infection process appears to conform to a classic model involving a receptor (F-pilus) and a co-receptor (TolA). How the phage penetrates the outer membrane and the
Gly2 257406
pIII
N2 N1
N2 N1
F pilus
TolA
F pilus
Figure 4 Infection of E. coli by Ff bacteriophage. Infection is initiated by interaction of the N2 domain of pIII with an F pilus projecting from an E. coli cell. This interaction releases the N1 domain from its intramolecular interaction with N2, allowing it to bind to a discrete domain (D3) of the bacterial TolA protein. Subsequent steps are not yet characterized. The bacterial outer membrane is omitted from the diagram for clarityit is not yet clear how infecting phage particles penetrate this membrane.
underlying peptidoglycan layer is not known. Three Tol proteins (Q , R, and A), all integral cytoplasmic membrane proteins, are absolutely required for phage infection (22, 23). They mediate depolymerization of the phage coat proteins into the cytoplasmic membrane and the translocation of the viral ssDNA into the bacterial cytoplasm, although the molecular details of how this is accomplished remain to be determined.
2.4 Replication
Upon entry of the viral single-stranded phage DNA (the strand, which has the same polarity as the mRNA), host RNA and DNA polymerases and topoisomerase convert it to a double-stranded, super-coiled molecule called Replicative Form (RF; see Figure 1). The RF serves as template for phage gene expression, and this expressionin particular, synthesis of pII, a site-specic nickingclosing enzymeis necessary for further replication. Through a rolling circle mechanism, pII nicks the strand of the RF at a specic site in the non-coding IG region of the phage genome, and the 3H end of the nick is elongated by host DNA polymerase III using the strand as template. The original strand is displaced by Rep helicase as the new strand is synthesized, and when a round of replication is complete, the displaced strand is recircularized by the nickingclosing activity of pII and again converted to RF. Synthesis of the strand requires an RNA primer. The primer is generated by RNA polymerase, which initiates synthesis at an unusual site in the IG region of the strand consisting of two adjacent hairpins that include promoter-like 35 and 10 motifs separated by a singlestranded region (24). During the early phase of infection, when the concentration of the phage ssDNA-binding protein (pV) is low, newly synthesized single strands are immediately converted to RF, and both RF and phage proteins increase exponentially. As its concentration increases, pV binds cooperatively to newly 6
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY generated strands (Figure 1), preventing polymerase access and blocking their conversion to RF. The restart protein pX, which is identical to the carboxy-terminal 111 residues of pII, is required for the stable accumulation of single strands at this stage, but the mechanism by which it acts is not known (25). pV is dimeric, with the interaction surface of the subunits opposite the DNA-binding surface. Thus upon binding, the back-to-back arrangement of the dimers collapses the circular single strand into a rod-like structure. The DNA is oriented in the complex, with the PS hairpin protruding from one end, presumably because pV bind dsDNA only weakly. The pV/ssDNA complex is the substrate for phage assembly.
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
II
............................................................................................................................................................................................................................................................................................................
II
............................................................................................................................................................................................................................................................................................................
111
III V
............................................................................................................................................................................................................................................................................................................
III V
406a 405 87
a
Virion component
Yes (N-term)
IV VI
............................................................................................................................................................................................................................................................................................................
IV
Assembly (exit channel) Outer membrane Cytoplasm Virion tip (end) Virion lament
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
VI
112 33
Virion component
Yes (C-term)
VII IX
a
............................................................................................................................................................................................................................................................................................................
VII IX
Virion component
VIII
............................................................................................................................................................................................................................................................................................................
VIII
50* 32
Virion component
Virion component
MARJORIE RUSSEL ET AL. promoters (there are only two terminators) and multiple RNA processing events increase the abundance of RNAs for the genes closest to the terminators (26). This results in high levels of pV and pVIII, the proteins that are required in the greatest quantities. At later times after infection, the rates of phage protein and DNA synthesis taper off. This occurs when the high concentration of pV sequesters the strands and prevents their conversion to RF, the template for gene expression. In addition, excess pV binds to a tetraplex structure in the gene II and gene X mRNAs (27), repressing their translation, and the reduction in pII levels leads to lower rates of strand synthesis (25, 28). At these lower synthetic rates, a steady-state level of phage products is maintained by the secretion of progeny phage and by the continued growth and division of the infected cells, and phage production continues at a linear rate.
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY cloned often give rise to smaller and/or clearer plaques; this may be due to the imbalance caused by the increased utilization of major compared to minor coat proteins in such particles.
MARJORIE RUSSEL ET AL. Elongation involves the successive replacement of pV dimers that cover the viral DNA by membrane-embedded pVIII and translocation of the DNA across the membrane. The process continues until the end of the viral DNA has been coated by pVIII. If either pIII or pVI is absent, the largely extracellular phage particle remains tethered to the cytoplasmic membrane where it remains competent to resume elongation when another pVssDNA complex enters the assembly site; ultimately, tethered phage laments of more than 10 times unit length accumulate (39). Even in normal infections when pIII and pVI are present, about 5% of progeny phage particles are double length. Such secondary rounds of elongation do not require reinitiationssDNA without a PS can be efciently incorporated (40). Pretermination (13) is the incorporation of the membrane-embedded pIIIpVI complex at the terminal end of the nascent phage particle. A fragment containing only the C-terminal 83 residues of pIII is sufcient to mediate this step, but cannot effect detachment of the phage from the cell. Termination or release of the phage, which requires a 93 residue C-terminal segment of pIII, has been proposed to consist of a conformational change in the pIIIpVI complex that detaches the complex (and the phage) from the cytoplasmic membrane (13). A still longer portion of pIII (the 132 C-terminal residues) is required for the formation of stable virus particles.
3.1 pVIII
pVIII, the major coat protein, is present in several thousand copies in phage particles. Sequences for display are typically inserted at the N-terminus, between the signal sequence and the beginning of the mature protein coding sequence. However, only short peptides sequences (68 residues) can be displayed on every copy of pVIII in a virionlarger sizes prevent packaging of the particles, probably because of the size restrictions of the pIV channel through which phage pass during extrusion (see Section 2.7). Display of larger polypeptides on pVIII 10
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY requires expression of the fusion protein from a phagemid vector, yielding hybrid virions bearing mainly wild-type pVIII (see Section 4.2). An emerging trend (for pVIII display but potentially more generally) is the use of protein engineering to modify the protein to broaden applicability: engineered pVIII proteins have been described that permit the display of large polypeptides at high copy number (42), or the display of proteins fused at the C-terminus of the protein (43).
3.2 pIII
pIII, present in ve copies at the ``end'' tip of the virion, is the protein of choice for most phage display fusions due to its tolerance for large insertions, its compatibility with monovalent display (see Section 4.3), and the wide availability of suitable vectors. Although pIII is more tolerant than pVIII to substantial insertions, infectivity of the resulting phage can be reduced, sometimes dramatically. As with pVIII, this can be overcome by using phagemid constructs, resulting in the production of hybrid virions that also bear wild-type pIII (see Section 4.2). Since such virions no longer rely on the infectivity of the pIII fusion protein, proteins can instead be fused to truncated pIIIs designed with the structure of the protein in mind (Figure 3). These can confer more efcient display, by reducing or eliminating proteolysis of the fusion protein, as well as reducing the size of the phagemid vector. Potential disadvantages include the possibility of sterically hindering access to the displayed protein. Fusions have been reported at pIII residue 198 (44), which deletes the N1 domain and some of N2, or to residue 249 (45), which deletes N1 and N2 and fuses the displayed protein to a short portion of the second glycine-rich linker. C-terminal pIII display through fusion to a linker at the C-terminus of the pIII is also possible (46).
............................................................................................................................................................................................................................................................................................................
Mustard trypsin inhibitor (MTI-2) Insulin-like growth factor (IGF)-1 Lipocalins hGH Trypsin
............................................................................................................................................................................................................................................................................................................
pIII
(48)
............................................................................................................................................................................................................................................................................................................
pIII (FosJun)
............................................................................................................................................................................................................................................................................................................
14.5 22 24 28 31 41
pIII
............................................................................................................................................................................................................................................................................................................
17.5
pIII
............................................................................................................................................................................................................................................................................................................
pIII, pVIII, pVIII (C-terminal) pIII, pVIII pIII, pVIII pIII pIII pIII, pVIII
(4244) (53)
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
25
(54)
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
Peptideb2mMHC complex Vascular endothelial growth factor (VEGF) Antibody Fab fragments Alkaline phosphatase Streptavidin
............................................................................................................................................................................................................................................................................................................
2 11.5 2 25 2 60 4 15 4
(59) (60)
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
(61)
............................................................................................................................................................................................................................................................................................................
(42)
............................................................................................................................................................................................................................................................................................................
(62)
c
Src Homology 3 (SH3) domain FRAP/mTOR FRB domain Zif268 zinc nger Cytochrome b562
............................................................................................................................................................................................................................................................................................................
6.5
pIII
(63)
d
............................................................................................................................................................................................................................................................................................................
9.5
pIII
............................................................................................................................................................................................................................................................................................................
10
pIII
64
............................................................................................................................................................................................................................................................................................................
11
pIII
(65)
............................................................................................................................................................................................................................................................................................................
12
pIII, T7 display
(66)
2 25.5
pIII
(67)
This table is intended to indicate the range of proteins that can be displayed, but is neither complete nor exhaustive. Additional examples can be found in (2). See Chapter 13 for additional references. T. Clackson, unpublished data. See Chapter 9 for additional references.
b c d
during preparation of phage displaying zinc nger proteins (see Chapter 9). For proteins with lone cysteine residues, such as FKBP12, selections can be performed in the presence of dithiothreitol (DTT) (to which phage particles are stablesee Section 5.2) to maintain a reducing environment (T.C., unpublished data). If necessary, displayed proteins can even be refolded prior to selection by exposure of the particles to denaturants followed by dialysis (68). It has also proved feasible to display multi-subunit proteins, by a variety of methods (Table 2). For heterodimers, such as antibody Fab fragments, one chain 12
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY can be displayed and another provided as a soluble protein cosecreted into the periplasm (60). Homo-oligomeric proteins can also be displayed by relying on proteolysis of the displayed fusion protein to release sufcient soluble protein, or by invoking interactions between displayed proteins, either inter- or intra-phage (e.g. see 61).
Phage vector
Ff ori
Ff ori
E. coli
(Defective Ff ori)
Polyvalent phage
Monovalent phage
Figure 5 General scheme for phage display using phage or phagemid vectors. The difference between phage and phagemid vectors is illustrated for pIII display. Sequences for display are inserted between a secretion signal sequence (``sig.'') and gene III. Both phage and phagemid vectors carry an Ff origin of replication to permit production of ssDNA and hence virions. Phagemid vectors also have a plasmid origin (here pBR322) and an antibiotic resistance marker to allow propagation as plasmids in E. coli. Phage vectors are also often modied with antibiotic resistance markers for convenience, as illustrated here. In many phagemid vectors, an amber stop codon (TAG) is interposed between the displayed sequence and gene III, to allow soluble protein expression by transferring the vector into a non-supE suppressor strain.
13
MARJORIE RUSSEL ET AL. another coat protein. When introduced into E. coli, phage will be produced in which all copies of the coat protein display the heterologous protein (i.e. the protein is displayed polyvalently). Examples of pIII phage display vectors include the fUSE vectors constructed by Smith and coworkers (69), and the M13KE vectors commercially available from New England Biolabs (see Section 8). In phagemid vectors, the displayed protein fusion gene is cloned into a small plasmid under the control of a weak promoter. In addition to a plasmid origin of replication, the vector also has an Ff origin to allow production of single-stranded vector and subsequent encapsidation into phage particles. To produce such particles, E. coli cells harboring the plasmid are infected with helper phage, which is an Ff phage with a compromised origin that leads to its inefcient packaging (see Section 4.4). The infected cells express all the wild-type phage proteins from the helper phage genome, as well as a small amount of the fusion protein encoded by the phagemid, so that phage particles are extruded by the cells that contain both proteins, usually with the wild-type in considerable excess. Because the helper phage genome is poorly packaged, nearly all the phage particles contain the phagemid genome, preserving the linkage between the displayed protein and its gene. Phagemid vectors have been described for both pIII and pVIII display, although pIII is more common. pIII phagemid vectors are described in more detail in Chapter 4. A major advantage of phagemid vectors is their smaller size and ease of cloning, compared to the difculties of cloning in phage vectors without disrupting the complex structure of overlapping genes, promoters, and terminators. This generally translates into much higher library sizes for phagemid vectors. In addition, the phagemid approach must be used if monovalent display is desired in order to obtain selection based on true binding afnity (see Section 4.3). For pVIII display, use of phagemids is generally required to achieve display of sequences longer than 68 amino acids. Phagemids have also been used for pVI display (see Chapter 12).
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY majority of the particles will display only wild-type pIII. Thus, the major displaying species is monovalent, but most particles do not display at all. The valency of display is important principally because of its impact on the ability to discriminate binders of differing afnities (see Chapter 4, Section 2.1). Early work (44) showed that polyvalent display prevented the highest-afnity clones in a selection from being identied, because multivalency conferred a high apparent afnity (avidity) on weak-binding clones. Monovalent display allows selection based on pure afnity, and is therefore generally preferred for the many studies where the aim is to identify the tightest binding variant(s) from a library. Conversely, in applications where the initial selectants are of very low afnityfor example, the de novo selection of peptides that bind a given targetpolyvalency increases the chances of isolating rare and weakly binding clones. A frequent experimental strategy in such projects is to start with polyvalent display, and then move to monovalent display as the afnity of the displayed polypeptide matures (see Chapter 6).
MARJORIE RUSSEL ET AL. coefcient depends on the size of phage particles, which in turn depends on the size of the genome (see Section 2.2). In general, for a helper phage such as VCSM13, 1 OD270 5 1012 particles/ml, and for a 5 kb phagemid, 1 OD270 1.1 1013 particles/ml. However, calculations based on absorbance may overestimate the concentration of viable, infective particles.
15 g agar; autoclave and cool to 55 C; pour into petri plates) 2YT top agar (16 g bacto-tryptone; 10 g bacto-yeast extract; 5 g NaCl; 7.5 g agar; add water to 1 L. Heat to dissolve the agar and autoclave.) Sterile 10 ml culture tubes Sterile Pasteur pipettes (for propagation) Microwave 45 C incubator or water bath (optional)
3 4 5
16
6 7
To each tube, add 3 ml of top agar, rolling the tube quickly to mix. Immediately pour the top agar mixture onto the surface of an LB agar plate, rotating the plate to spread the top agar quickly and evenly across the surface. If the top agar is too cool, lumps will form, making plaques difficult to count. Allow the top agar to solidify, then incubate the plates at 37 C overnight. Plaques should appear as relatively clear discs against a background lawn of cells.
8 9
10 Calculate the phage concentration in the original stock solution (plaque-forming units/ml; pfu/ml) by multiplying the number of plaques per plate by 100 (because 0.01 ml of diluted phage is used per plate) and by the appropriate serial dilution factor.
B. Progagation
1 To propagate phage, grow fresh XL1-Blue cells as above, and pick a single isolated plaque using a Pasteur pipette by placing a finger over the open pipette end after inserting it through a plaque into the agar. Using a pipette bulb, force the agar plug into 1 mL of fresh XL1-Blue cells.
3. Incubate for 1 h at 37 with shaking or rotation. 4. Dilute the cells into 251000 ml of 2YT media containing an appropriate antibiotic (e.g. 10 mg/ml kanamycin for M13K07 or M13VCS helper phage). 5 Incubate the culture for 1215 h at 37 C with shaking. Phage may be harvested and purified as described in Chapter 2, Protocol 6.
This procedure is a modification of that described by Sambrook et al. (72) for plating M13 phage. A typical yield from XL1-Blue phage cultures grown in 2YT would be 10111012 phage/ml.
General principles: Ch. 4 Oligo-directed mutagenesis: Ch. 2 In vitro recombination: Ch. 3 Oligo cassette: Ch. 6 Recursive PCR: Ch. 8 Oligo-splinted assembly: Ch. 9 Error-prone PCR: Ch. 11 PCR with randomized primers: Ch. 14
Premade peptide libraries: Ch. 1 sect. 7 Gene fragment libraries: Ch. 11 cDNA libraries: Ch. 12 Antibody V-region libraries: Ch. 13
Select
General principles: Ch. 4 In vitro binding: Chs 4, 6, 9, 11, 12, 13 Enzymatic selection: Chs. 7, 8 In vivo binding: Ch. 10
Sequencing: Ch. 4 sect. 7 PCR fingerprinting: Ch. 13 Binding, by phage ELISA: Ch. 5 (also see Chs. 6, 9, 11, 12, 13) Test soluble proteins: Chs. 4, 6, 13, 14
Figure 6 Flow diagram illustrating a generic phage display project, and examples of techniques that can be used at each stage (boxed).
recent advances have brought larger libraries of 10101011 clones within easy reach (see Chapter 2). The diversity introduced can involve complete (``hard'') randomization of residues, partial (``soft'') randomization in which the wild-type residue is retained in some proportion, or ``tailored'' randomization, in which only a dened subset of amino acids is specied (see Chapter 2, Section 2, and Chapter 4, Section 3; for an example of tailored randomization, see Chapter 8). In all targeted libraries, it is important to bear in mind the implications of library size limitations on the total amount of diversity that can be surveyed in one library (see Chapter 2, Section 2). Alternatively, introduction of random changes throughout a sequence can be accomplished by in vitro recombination (Chapter 3). Numerous alternative approaches for introducing targeted diversity are described in other chapters throughout the book, as indicated in Figure 6. Some specialized phage display applications involve the creation of libraries from collections of genes as opposed to diversifying a single gene. Examples 18
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY include cDNA libraries (Chapter 12) and antibody V-region libraries (Chapter 13). In these cases, specic protocols have been developed to create these libraries. The creation of a library can be completely circumvented in some cases by obtaining an appropriate pre-made phage display library. Pre-made peptide display libraries are now commercially available (see Section 8).
5.2 Selection
The most common means of selection of desired clones is through an in vitro binding incubation, in which the phage library is bound to a target, washed, and then retained phage are eluted. This process is also referred to as ``sorting'' or ``biopanning.'' The principles and alternatives for selections based on in vitro binding are described in detail in Chapter 4, Sections 4 and 5. A wide variety of washing and elution conditions have been used, exploiting the extraordinary stability of the Ff phage virion to extremes of pH, ionic strength, denaturants and even most proteases (with the exception of subtilisin) (see Table 3). This stability means that selection approaches are essentially limited only by the imagination of the investigator: for example, selection is even possible based on binding to targets in vivo after injection of phage libraries into whole animals (see Chapter 10). The enrichment for binding clones over nonbinders conferred by a single round of selection can vary widely, from two-fold to more than a 1000-fold, although at least 10-fold is typical for an in vitro binding selection. In most cases, the eluted phage are used to reinfect E. coli for preparation of new phage (``amplied''), to allow further rounds of selection. Enrichment is typically monitored to help guide the decision to stop selecting and start analyzing clones.
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................
(77)
............................................................................................................................................................................................................................................................................................................
(78)
............................................................................................................................................................................................................................................................................................................
(79)
............................................................................................................................................................................................................................................................................................................
(80)
............................................................................................................................................................................................................................................................................................................
(45, 81)
19
MARJORIE RUSSEL ET AL. and considerations for when to stop selecting and start analyzing, are described in Chapter 4, Section 7. An alternative and rapid approach for clone identication is PCR ``ngerprinting,'' in which the insert encoding the displayed protein is amplied by PCR, and then digested with a frequent-cutting restriction enzyme (see Chapter 13, Protocol 19). Analysis of the digestion products gives a pattern of bands that may be unique to that clone. PCR ngerprinting techniques are particularly useful in applications such as cDNA library or phage antibody selections, where each clone is likely to have a different restriction pattern. The most popular and versatile technique for initial characterization of the properties of selected clones, particularly for the majority of studies based on binding selections, is the phage ELISA. In this method, the target of interest is immobilized in wells of a 96-well plate, individual phage supernatants prepared from selected clones are added, and specic binding is detected by use of an anti-phage antibody. In addition to giving ``yesno'' information as to whether a given clone binds the target, the assay can be used in a competition format to determine the relative binding afnities of clones, as described in Chapter 5. Following characterization of the displayed polypeptide ``in situ'' on phage, the next step is often the production of soluble (undisplayed) protein for more in-depth analysis. For most monovalent display vectors this is facilitated by the presence of an amber stop codon between the displayed protein and pIII, allowing soluble non-fusion protein to be expressed alone by transfer to a non-suppressor strain of E. coli (45, 60) (see Figure 5). Often the expression and subsequent binding analysis can be performed at small scale in 96-well plates (e.g. see Chapter 13). For shorter peptide sequences, expression as fusions to wellcharacterized afnity proteins such as maltose binding protein (MBP) can allow conrmation of the properties observed for the displayed peptide (see Chapter 6). With efcient procedures available for library generation and selection, downstream analysis of clones can frequently be the rate-limiting step in a phage display project. This is leading to the increasing use of automation in an effort to improve throughput: for example, the use of Q-pix and Q-bot robots for colony picking is described in Chapter 11.
6 Common problems
Many chapters in this book contain troubleshooting hints for specic aspects of phage display, or for particular applications: for example, a guide for phage display binding selections is provided in Chapter 4 (Table 3). However, there are several more general potential pitfalls and problems to avoid that can be identied.
INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY to lead to success. It is worth investing the time to ensure and then conrm the quality and diversity of the starting libraryfor example, by using a template encoding an inactive protein when creating a library using oligonucleotide-directed mutagenesis (see Chapter 2, Section 3.3).
6.3 Over-selection
A common observation in monovalent display, but one that understandably often fails to appear in publications, is that apparently successful selections can often yield clones with strange and unexpected structures, or those that upon analysis do not have the property selected for (e.g. higher afnity). In many cases these outcomes can be attributed to over-selection of the library beyond the rounds at which the desired clones are dominant. Under these conditions, the imposed selective pressure for binding afnity (for example) becomes ineffective, since nearly all clones at that stage will be of equivalent afnity, and factors such as expression level and valency start to drive selection. The result is often that bizarre clones are selectedfor example those with internal duplications that lead to bivalency, or those of weak afnity but which are displayed at very high levels. Such observations are a testimony to the power of selection techniques, but emphasize that selections should be carefully monitored, and samples from each round of selection preserved for future analysis.
MARJORIE RUSSEL ET AL. disulde formation provides a C-terminally displayed protein system that has been useful for cDNA interaction cloning. A second technique, termed selectively-infective phage (SIP), exploits the modularity of pIII (see Figure 3) to establish a ``two-hybrid''-related system in which pIII is split into two pieces, rendering the phage noninfectious. A high-afnity interaction between proteins fused to the pIII domains restore infectivity, allowing identication of binding partners without the need for afnity selection (85). In addition, bacteriophages other than the Ff family have been used for display of polypeptides, including the lambda (86), T4 (87), and T7 phages (e.g. see (88) ). Display as C-terminal fusions to the gene 10 capsid protein of phage T7, in particular, is being increasingly used due to the availability of commercial kits and libraries (see Section 8). Because these phage are lytic, in many cases display does not involve the secretion of the fusion proteins, potentially conferring an advantage for display of some normally intracellular proteins (although see Section 4.1). An application of T7 display phage is described in Chapter 10. Lytic phage are also useful for cDNA display (see Chapter 12, Section 2.2). Finally, alternative platforms for in vitro selection outside of phage display include display on ribosomes following arrest of translation, and display on DNA binding proteins such as the lac repressor (for a review, see (89)). In some cases these techniques have been used together with phage display in a single project aimed at discovering and then improving new binding molecules (see Chapter 6).
Table 4 Commercial suppliers of phage display vectors and libraries Tradename PhD
TM
Manufacturer phage display system M13KE pIII phage vector Pre-made peptide libraries with control target and eluant; starting phage vector
Vector
Reagents available
Website www.neb.com
................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................
Amersham Biosciences
................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................
Kits for library construction, selections, phage enzyme-linked immunosorbant assays (ELISAs), and expression of soluble proteins; starting phagemid vector
www.amershambiosciences.com
Novagen
T7Select1 System
Kits for library construction, phage in vitro packaging, and selections; pre-made cDNA display libraries
www.novagen.com
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