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WO2005062045A1 - Procede pour detecter une interaction entre un polypeptide et un recepteur, procede pour cribler un ligand ou un variant de ligand au moyen de ce procede de detection, et procede de diagnostic utilisant ledit procede de detection - Google Patents

Procede pour detecter une interaction entre un polypeptide et un recepteur, procede pour cribler un ligand ou un variant de ligand au moyen de ce procede de detection, et procede de diagnostic utilisant ledit procede de detection Download PDF

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Publication number
WO2005062045A1
WO2005062045A1 PCT/JP2004/018347 JP2004018347W WO2005062045A1 WO 2005062045 A1 WO2005062045 A1 WO 2005062045A1 JP 2004018347 W JP2004018347 W JP 2004018347W WO 2005062045 A1 WO2005062045 A1 WO 2005062045A1
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WIPO (PCT)
Prior art keywords
receptor
polypeptide
ligand
carrier
membrane
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PCT/JP2004/018347
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English (en)
Japanese (ja)
Inventor
Masaharu Seo
Tadahiro Nagaoka
Hiroshi Okamura
Kouichi Hirayama
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Toyo Kohan Co., Ltd.
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Publication of WO2005062045A1 publication Critical patent/WO2005062045A1/fr

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    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • a method for detecting the interaction between a polypeptide and a receptor a method for screening a ligand or a ligand variant using the method for detection, and a diagnostic method using the method for detection
  • the present invention relates to a method for detecting an interaction between a protein and a membrane receptor, a method for screening a ligand or a ligand variant using the detection method, and a diagnostic method using the detection method.
  • Receptors are fundamental biomolecules involved in the development, maturation, growth, metabolism, maintenance, etc. of life, and the enormous interest and efforts of researchers have led to the structure, function, mechanism, mechanism of such receptors, etc. Has been poured into.
  • many chemicals have been screened using receptors as targets for therapeutic drugs and pesticides.
  • Receptors are classified into membrane receptors, nuclear receptors, transporters, cofactors, coactivators, etc. according to their functions and structures.
  • the types of proteins reported as these receptors have been enormous in recent years and continue to increase. More than 100 types of receptors called nuclear receptors have been reported.
  • the number of receptors reported as membrane proteins existing on or in the biological membrane is enormous.
  • Non-Patent Document 1 Screening for polypeptides that interact with these receptor proteins is important for finding effective drugs.
  • Methods for detecting the interaction between specific proteins include immunoprecipitation and Western blotting, but these methods are not suitable for comprehensive analysis.
  • Yeast Two-Hybrid analysis can perform comprehensive analysis. Many false positives and false negatives are detected, and it is difficult to immediately determine true positives and true negatives.
  • Non-Patent Document 2 protein chips have been developed using DNA array technology representing a comprehensive analysis method as a tool for protein analysis. The principle of a tin chip is the same as that of a DNA chip, in which proteins are immobilized at high density on a slide glass or membrane, and proteins and nucleic acids that interact with them are detected.
  • membrane receptors present on the cell membrane surface have highly hydrophobic transmembrane domains, so that they are difficult to dissolve.
  • Patent Document 1 JP-A-10-174591
  • Non-patent Document 1 Edited by Tadaomi Takebashi et al., Bio Manual UP Series, Experimental Method for Intermolecular Interaction of Proteins, Yodosha, 1996
  • Non-Patent Document 2 Lam KS et.al, Nature 354, 82 (1991)
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have prepared a receptor derivative containing an extracellular domain in a membrane receptor but not a region essential for membrane binding. was reacted with a polypeptide immobilized on a carrier, and it was found that the above problem could be solved by detecting the interaction between the two, and the present invention was completed.
  • the present invention includes the following inventions.
  • a polypeptide is immobilized on a carrier, and a ⁇ ⁇ receptor derivative containing an extracellular domain of a membrane receptor but not containing a region essential for membrane binding is reacted with the polypeptide, and the immobilized polypeptide and the receptor derivative are reacted.
  • a method for detecting an interaction between a polypeptide and a receptor by detecting an interaction between the polypeptide and the receptor.
  • the carrier is a solid support having a functional group capable of covalently bonding to the polypeptide on the substrate and at least one surface layer selected from diamond, soft diamond, carbon-based materials and carbides. ) Described method.
  • FIG. 1 is a diagram schematically showing the structure of ErbB extracellular domain (ECD) -hinge-FLAG.
  • FIG. 2 shows the amino acid sequence of ErbB1 ECD-hinge-FLAG and the amino acid sequence of ErbB4 ECD-hinge-FLAG (top), and the amino acid sequence of betacellulin having an epitope tag at the C-terminal and N-terminal (bottom). It is a figure showing structurally.
  • FIG. 3 shows Example 5, which is a purified recombinant human betacellulin immobilized on a carrier (left), a crude extract containing Myc-betacellulin HA (center), and a crude extract containing a control vector. (Right) is a fluorescence image taken by interacting the biotinylated ErbBl ECD-hinge-FLAG and further reacting with Cy3-avidin.
  • Fig. 4 shows the relationship between the recombinant betacellulin concentration and the fluorescence intensity in each spot in the fluorescence image of Fig. 3 (left), the dilution ratio and fluorescence of the crude extract containing Myc-betacellulin HA in each spot. It is a figure showing the relationship with intensity (right). NC stands for control vector.
  • Figure 5 shows the combination of a biotinylated ErbB ECD-hinge-FLAG and avidin-Cy3-Piotin BSA It is a figure showing interaction with a body.
  • the membrane receptors targeted by the present invention include receptors for peptide hormones, neurotransmitters, growth factors, cytokins, catecholamines, etc. Functionally, trimer G protein-coupled receptors, ion channel conjugates Type receptors, protein kinase type receptors, and co-receptors. It can also be classified into receptors that penetrate the plasma membrane once, receptors that penetrate the plasma membrane four times, receptors that penetrate the plasma membrane seven times, and GPI-anchored receptors that bind to the membrane via lipids. INDUSTRIAL APPLICABILITY The present invention is suitable for detecting an interaction between a polypeptide and a receptor that once penetrates the plasma membrane or a GPI-anchored receptor.
  • EGF receptor EGF receptor, PDGF receptor, adenosine receptor, FGF receptor, TGF jS receptor, insulin receptor, IGF-I receptor, angiotensin receptor, OB receptor, melanocortin receptor, adrenergic receptor, thrombin receptor, oxytocin receptor, Inositol triphosphate receptor, FSH receptor, TSH receptor, interferon receptor, interleukin receptor, G-CSF receptor, chemokine receptor, tyrosine kinase receptor, GDNF receptor, TNF receptor, glypican and the like.
  • the present invention is suitably used for EGF receptors.
  • EGF receptor As an EGF receptor, a EGF receptor,
  • ErbBl, ErbB2, ErbB3, and ErbB4 can be mentioned, and particularly preferably used for ErbBl and ErbB4.
  • the membrane receptor contains a region essential for membrane binding and an extracellular domain present extracellularly and interacting with a ligand.
  • the region essential for membrane binding means a region in a membrane receptor that is responsible for binding between the membrane receptor and a cell membrane. Such regions include, but are not limited to, transmembrane domains that are usually composed of hydrophobic amino acids, and regions that contain amino acids that bind GPI anchors that bind to membranes through lipids. is not.
  • a receptor derivative refers to a derivative of a membrane receptor that contains an extracellular domain of a membrane receptor but does not contain a region essential for membrane binding. Below receptor One derivative may be referred to as a soluble receptor derivative.
  • the receptor derivative of the present invention is soluble in water because it does not contain a region essential for hydrophobic membrane binding. Since the receptor derivative contains an extracellular domain that interacts with the ligand, the reactivity with the ligand is maintained. From the viewpoint of maintaining solubility, the receptor derivative of the present invention is preferably a membrane receptor component that contains only a domain essential for affinity with a ligand and does not contain any other domain.
  • Receptor mono-derivatives can be prepared by methods commonly used in the art based on DNA encoding a known membrane receptor. That is, it can be prepared by inserting DNA encoding the receptor derivative into a vector, transforming a host cell with the vector, and culturing the transformant.
  • the receptor derivative of the present invention also includes a polypeptide obtained by fusing another peptide to a polypeptide which contains an extracellular domain of a membrane receptor but does not contain a region essential for membrane binding.
  • Other peptides to be fused include, for example, FLAG (Hopp, TP et.
  • 6 X His consisting of 6 His (histidine) residues, 10 X His, Human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, a-tubulin fragment, B-tag, Protein C Fragments, GST (glutathione S-transferase), HA (influenza agglutinin), immunoglobulin constant region, immunoglobulin hinge region, j8-galactosidase, MBP (maltose binding protein) and the like.
  • the receptor derivative of the present invention preferably has an immunoglobulin hinge region fused thereto, whereby the receptor derivative can be dimerized by an SS bond. In many cases, by dimerization, the binding property to the polypeptide on the carrier is increased, so that the detection sensitivity can be improved.
  • the immunoglobulin hinge region is fused to the extracellular domain of the membrane receptor, and further an epitope tag, particularly a FLAG-tag. The presence of the immunoglobulin hinge region promotes dimerization, and the presence of the FLAG-tag facilitates purification of the receptor derivative and detection of interaction with the polypeptide.
  • a vector used for preparing a receptor derivative a vector generally used in the art can be used.
  • E. coli when E. coli is used as a host in transformation, for example, ⁇ ori '' for replication in E. coli and a gene for selecting transformed E. coli (for example, ampicillin, tetracycline,
  • a vector which desirably has a drug resistance gene such as namycin or chloramuecole) on a vector is specifically exemplified by M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and pGEM-T. , PDIRECT, pT7 etc.
  • an expression vector is useful.
  • expression vectors for example, when expression in E. coli is intended, lacZ promoter (Ward et. Al., Nature 341: 544-546, 1989) and araB promoter (Better et. Al., Science 240: 1041- 1043, 1988), or a vector having a T7 promoter or the like.
  • Such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, and pET (in this case, the host is BL21 expressing T7 RNA polymerase)
  • the expression vector may contain a signal sequence for polypeptide secretion.
  • vectors include, for example, mammalian-derived expression vectors (eg, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 18 (17): 5322, 1990, pEF, pCDM8), Expression vectors derived from insect cells (eg, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8), expression vectors derived from plants (eg, ⁇ 1, pMH2), expression vectors derived from animal viruses (eg, pHSV , PMV, pAdexLcw), a retrovirus-derived expression vector (eg, pZIPneo), a yeast-derived expression vector (eg, “Pichia ExpressionKit” (manufactured by Invitrogen), pNVll, SP-Q01), an expression vector derived from Bacillus subtilis ( For example, pPL608, pK
  • a promoter necessary for expression in cells for example, the SV40 promoter (Muligan et al., Nature 277: 108) , 1979), MMLV-LTR promoter, EF1 promoter (Mizushima et. Al, Nucleic Acids Res. 18: 5322, 1990), CMV promoter, etc. It is indispensable to have a gene (for example, a drug (neomycin, G418, etc.) resistance gene) for selecting for cell transformation.
  • Vectors having such properties include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13 and the like.
  • the replication origin those derived from poliovirus, adenovirus, dysapilovirus (BPV) and the like can also be used.
  • the expression vector is used as a selectable marker for aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt) gene for gene copy number amplification in host cell systems. And a dihydrofolate reductase (dhfr) gene.
  • the host into which the vector is introduced is not particularly limited, and for example, Escherichia coli and various eukaryotic cells can be used.
  • eukaryotic cells for example, animal cells, plant cells, and fungal cells can be used as hosts.
  • Animal cells include mammalian cells, for example, CHO, COS ⁇ 3T3, myeloma, BHK (baby hamster kidney), HeLa ⁇ Vero, amphibian cells, for example, African omega oocytes (Valle, et. Al., Nature 291: 358-340, 1981), or insect cells such as S19, Sf21, and Tn5.
  • CHO cells in particular, DHFR-deficient CHO cells such as dhfr-CHO (Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980) and CHO K-1 (Proc. Natl. Acad. Sci. USA 60: 1275, 1968).
  • CHO cells are particularly preferred for the purpose of large-scale expression in animal cells.
  • the plant cells include cells derived from -Cotiana 'tapacum (Nicotiana tabacum).
  • yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae
  • filamentous fungi such as the genus Aspergillus, such as Aspergillus niger
  • bacterial cells include Escherichia coli (E. coli) such as JM109, DH5a, HB101, XLlBlue, BL21, and Bacillus subtilis.
  • mammals, plants, and insects may be used. Goats, pigs, sheep, mice, and horses can be used as mammals (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993).
  • tobacco can be used.
  • silkworms can be used as insects, for example.
  • the above-mentioned vector is introduced into the above-mentioned host.
  • the calcium chloride method and the elect-portion method (Chu, G. et. Al., Nucl. Acid Res. 15: 1311-1326, 1987) ) Can be used.
  • the calcium phosphate method Choen, C. and Okayama, H. Mol. Cell. Biol. 7: 2745-2752, 1987
  • the DEAE dextran method Lipata, MA et. Al, Nucl. Acids Res. 12: 5707-5717, 1984, Sussman, DJand Milman, G. Mol. Cell. Biol. 4:
  • the DNA when introducing DNA into an animal, the DNA is incorporated into an appropriate vector (for example, an adenovirus vector (for example, pAdexlcw), a retrovirus vector (for example, pZIPneo), etc., but not limited thereto).
  • an adenovirus vector for example, pAdexlcw
  • a retrovirus vector for example, pZIPneo
  • it can be introduced into a living body by a retrovirus method, a ribosome method, a cationic ribosome method, an adenovirus method, or the like.
  • a vector when introduced into an insect, it can be carried out, for example, by infecting a silkworm with a baculovirus into which DNA encoding a protein of interest has been inserted (Susumu, Met. Al., Nature 315: 592- 594, 1985).
  • DNA is introduced into a plant
  • a DNA encoding a target protein is introduced into a plant expression vector, for example, pMON530, and this vector is used as a vector such as Agrobacterium tumefaciens. Introduce into bacteria.
  • the vector can be introduced by infecting the bacterium with tobacco, for example, -Cotiana 'tapacam (Julian K.-C. Ma et.al, Eur. J. Immunol. 24: 131-138, 1994).
  • the soluble receptor derivative according to the present invention can be obtained by culturing the above transformant. Can be produced.
  • the culture can be performed according to a known method.
  • DMEM, MEM, RPMI1640, IMDM, and the like can be used as a culture solution.
  • a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed.
  • FCS fetal calf serum
  • the pH during the culture is preferably about 6-8. Culture is usually performed at about 30-40 ° C for about 15-500 hours, and if necessary, the medium is replaced, aerated, and agitated.
  • the soluble receptor derivative according to the present invention can be isolated from the inside or outside of a host cell (such as a medium) and purified as a substantially pure and homogeneous polypeptide.
  • the separation and purification of the polypeptide is not limited at all, provided that the separation and purification methods used in ordinary polypeptide purification are used. For example, chromatographic columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. If combined, the polypeptide can be separated and purified. Alternatively, it is possible to purify further by combining a plurality of these columns.
  • chromatography examples include affinity chromatography in which an antibody against the above-mentioned epitope tag is immobilized on a column, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like. (Strategies for Protein Purification ana Characterization: A Laboratory and our Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996).
  • the soluble receptor derivative of the present invention may be used as a fusion protein with daltathione S-transferase protein, or as a recombinant protein to which a plurality of histidines have been added, in host cells (eg, animal cells, Escherichia coli, etc.).
  • host cells eg, animal cells, Escherichia coli, etc.
  • the expressed recombinant protein can be purified using a daltathione column or a nickel column. After purification of the fusion protein, if necessary, a region other than the target protein in the fusion protein can be cleaved with thrombin or Factor Xa and removed.
  • the polypeptide is immobilized on a carrier, and the polypeptide is soluble in the polypeptide.
  • the butter derivative is reacted.
  • the polypeptide immobilized on the carrier may be any polypeptide that has a possibility of interacting with the membrane receptor. It may be a naturally occurring polypeptide or an artificially synthesized one.
  • the polypeptide includes oligopeptides, proteins, protein fragments, and the like.
  • a ligand for a membrane receptor from which the soluble receptor derivative is derived by immobilizing an unknown polypeptide on a carrier and detecting the interaction with the soluble receptor derivative, that is, the presence or absence of binding. it can.
  • the extracellular domain is responsible for the interaction with the ligand, and the interaction with the original membrane receptor is detected by detecting the interaction with the soluble receptor derivative of the present invention containing the extracellular domain. Has the same effect as detecting.
  • a spotting solution is prepared by dissolving or dispersing the target polypeptide or a sample containing the polypeptide in a solvent. By incubating the spotting solution after spotting on the carrier, the polypeptide can be immobilized on the carrier.
  • the solvent for example, distilled water, SSC (saline-sodium citrate), PBS (phosphate
  • NaHCO sodium bicarbonate
  • One or more solvents such as dimethylsulfoxide, dimethylformamide, tetrahydrofuran, trifluoroacetic acid, triethylamine, 1-methyl-2-pyrrolidone, dioxane, and ethyl acetate can be used.
  • PBS polymethylsulfoxide
  • Spotting is performed by dispensing a spotting solution containing a polypeptide into a 96- or 384-well plastic plate, and dropping the dispensed solution onto a carrier using a spot device or the like.
  • a spot device a pin-type device is generally used in which a pin holds a sample solution, the pin is brought into contact with the carrier surface, and the solution is transferred to the carrier surface to form a spot.
  • pin tip shapes such as solid pin type (particularly, one with no groove) and tile pin type (one with a groove like a fountain pen). Can also be used. Preferably, it is a tile pin type.
  • an ink jet method using the principle of an ink jet printer and a spot device using a capillary method using a capillary are also used. be able to.
  • the spotting amount of the spotting solution per spot can be appropriately determined by those skilled in the art, but is usually in the range of lpL to 1 ⁇ L, preferably in the range of 100 pL to 100 nL. Spot sizes are usually in the range of 50-300 m in diameter. And the distance between the spots is usually in the range of 0-1.5 mm, preferably in the range of 100-700 m. After spotting, it is preferable to incubate the mixture at room temperature—50 ° C., preferably 30-42 ° C., usually for 3 hours or less, preferably 0.5-1.5 hours.
  • the carrier is washed to remove the polypeptide and the like that have not been immobilized.
  • the washing solution those commonly used in the art can be used.
  • an aqueous solution containing 2 ⁇ SSC, 0.2% SDS (sodium dodecyl sulfate) can be used. In this way, a carrier having hundreds of thousands of spots can be obtained.
  • the spotting solution preferably contains a polyhydric alcohol.
  • Polyhydric alcohol refers to an organic compound having two or more hydroxyl groups. Specifically, glycerin, saccharides (glucose, mannose, galactose, fructose, maltose, isomaltose, cellobiose, ratatose, sucrose, glycogen) , Amylose, cenorelose, trehalose, glycosides, dalconic acid, galactonic acid, galataturonic acid, mannonic acid, darconolatatone) and the like, with glycerin being particularly preferred.
  • Inclusion of polyhydric alcohol can prevent drying of the solution after spotting and improve the detection sensitivity by covalently binding the polypeptide to the substrate surface without inhibiting the chemical reaction in the aqueous solution It can be done.
  • the volume of the solution containing the polypeptide is low, so that the spot is easy to dry.
  • the concentration of the polyhydric alcohol contained in the spotting solution is usually 5 to 30% by mass, preferably 10 to 20% by mass.
  • the crude cell extract before purifying the polypeptide is dissolved as it is or after dissolving or dispersing it in the above-mentioned solvent, and the above-mentioned polyhydric alcohol is added thereto.
  • the polypeptide of the present invention can be immobilized on a carrier and the interaction with a soluble receptor derivative can be detected. That is, in the present invention, even when impurities other than the target polypeptide are mixed in the spotting solution, the interaction with the soluble receptor derivative can be detected.
  • a biological sample for example, urine, blood, serum, plasma, saliva, nasal discharge, tear fluid, cerebrospinal fluid, lymph, sweat, or the like is dissolved or dispersed in the above-mentioned solvent as it is,
  • the protein contained in the sample can be immobilized on a carrier as a spotting solution by adding a polyhydric alcohol to the solution, and the interaction with the soluble receptor derivative can be detected.
  • a carrier as a spotting solution by adding a polyhydric alcohol to the solution, and the interaction with the soluble receptor derivative can be detected.
  • it can be determined whether such a sample contains a substance that interacts with the receptor.
  • a variant of a ligand that binds to a receptor derived from a receptor derivative used is immobilized on a carrier as a polypeptide.
  • a ligand mutant is prepared by inserting a DNA encoding a ligand mutant into a vector based on a DNA encoding a known ligand, and transforming the host cell using the vector. It can be prepared by transforming and culturing the obtained transformant.
  • the purification method and the like can be performed in the same manner as the receptor derivative, but as described above, spotting is performed using a crude cell extract before purifying the ligand variant from the host cell culture.
  • EGF receptor / EGF EGF receptor / EGF
  • adenosine receptor / adenosine PDGF receptor / PDGF
  • FGF receptor / FGF TGF j8 receptor / TGF j8, insulin receptor / insulin
  • IGF-I receptor / IGF-I angiotensin receptor / angi OT
  • OB receptor / OB melanocortin receptor / melanocortin
  • adrenaline receptor / adrenaline thrombin receptor / thrombin
  • oxytocin receptor / oxytocin inositol triphosphate receptor / inositol triphosphate
  • FSH receptor / FSH TSH receptor / TSH
  • interferon receptor / interferon interleukin receptor / interleukin
  • G-CSF receptor / G-CSF chemokine receptor
  • ErbBl / EGF ErbBl / TGF- ⁇ , ErbBl / amphiregulin, ErbBl / epiregulin, ErbBl / heparin-binding EGF (HBEGF), ErbB3 / -euregulin 1, ErbB3 / -euregulin 2a, ErbB3 / -euregulin 2 ⁇ , ErbB4 / -Euregulin 2a, ErbB4 / -Euregulin 2 ⁇ , ErbB4 / -Euregulin 3, ErbB4 / -Euregulin 4, ErbB4 / HBEGF, and combinations of ErbB4 / Epiregulin.
  • HBEGF ErbB4 / Epiregulin.
  • the DNA encoding the ligand variant can be prepared by those skilled in the art using methods commonly used in the art, for example, site-directed mutagenesis (Hashimoto-Gotoh, T. et.al, Gene 152: 271-275, 1995, Zoller, MJ and Smith, M. Methods Enzymol. 100: 468-500, 1983, Kramer, W. et.al, Nucleic Acids Res. 12: 9441-9456, 1984, Kramer, W. and Fritz, HJ Methods. Enzymol. 154: 350-367, 1987, Kunkel, TA et. Al., Proc Natl Acad Sci USA. 82: 488-492, 1985, Kunkel et. Al "Methods Enzymol. 85: 2763-2766. , 1988) and the like.
  • site-directed mutagenesis Hashimoto-Gotoh, T. et.al, Gene 152: 271-275, 1995, Zoller, MJ and Smith,
  • another peptide may be fused to the ligand variant.
  • Introduction of mutation into ligand inhibits translation of ligand, etc. and transcribes to terminal * Power that may not be translated
  • an epitope tag such as FLAG-tag or HA to N-terminus or C-terminus of ligand variant It can be confirmed that the transcript is translated to the end.
  • Ligand variants can be screened.
  • betacellulin which belongs to the epidermal growth factor (EGF) family, is known to be a ligand that interacts with both ErbBl and ErbB4 of the EGF receptor family.
  • EGF epidermal growth factor
  • the betacellulin mutant interacts with ErbBl but does not interact with ErbB4, or interacts with ErbB4 but interacts with ErbBl.
  • Betacellulin mutants that do not act can be screened.
  • the detection method of the present invention can be used to screen for a drug that competitively binds to the receptor and inhibits or activates the original function.
  • the candidate conjugate or the ligand variant is spotted on a carrier, and the polypeptide contained in each sample is immobilized on the carrier to form a membrane receptor. By detecting the interaction of, the desired candidate substance can be rapidly and easily screened.
  • the carrier for immobilizing the polypeptide those commonly used in the art can be used.
  • noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductive materials such as graphite and carbon represented by carbon fiber; single-crystal silicon, amorphous Semiconductor materials typified by silicon, silicon carbide, silicon oxide silicon, silicon nitride, and the like; composite materials of these semiconductor materials typified by SOI (silicon 'on' insulator); glass, quartz glass, alumina, Inorganic materials such as sapphire, ceramitas, forsterite, and photosensitive glass; polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorinated resin, polychlorinated vinyl, polychlorinated vinylidene, and polystyrene Polyvinyl acetate, Polyvinyl alcohol, Polyvinyl acetate Ac
  • a carrier for immobilizing a polypeptide a functional group capable of covalently bonding to the polypeptide on a substrate, and at least one kind selected from diamond, soft diamond, a carbon-based substance, and a carbonide It is preferable to use a solid support having a surface layer. By using such a solid support, the polypeptide can be firmly immobilized and the unpurified polypeptide can be immobilized.
  • Examples of the material of the substrate used for the solid support include silicone, glass, fiber, wood, paper, ceramics, and plastics (eg, polyester resin, polyethylene resin, polypropylene resin, ABS resin). (Acrylonitrile Butadiene Styrene ⁇ fats), nylon, Acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, Shiridani vinyl resin), synthetic diamond, high-pressure synthetic diamond, natural Diamond, soft diamond (for example, diamond-like carbon), amorphous carbon; metals such as gold, silver, copper, aluminum, tungsten, and molybdenum; mixing and bonding of the above-mentioned resin to the above-mentioned metal powder, ceramic powder, etc. as a binder Formed: What is obtained by sintering at a high temperature a material obtained by pressing a raw material such as the metal powder or ceramic powder by a press molding machine.
  • plastics eg, polyester resin, poly
  • the solid support of the present invention has a surface layer on a substrate.
  • a compound for introducing a functional group capable of covalently bonding to the polypeptide can be firmly fixed on the substrate.
  • the surface layer is formed of at least one kind selected from diamond, soft diamond, carbon-based material and carbide.
  • the diamond, soft diamond, carbon-based material and carbide include synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (eg, diamond-like carbon), amorphous carbon, and carbon-based material (eg, graphite, fullerene, Carbon nanotubes), a mixture thereof, or a laminate thereof, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, And carbides such as chromium carbide and vanadium carbide.
  • soft diamond is a general term for imperfect diamond structures, which are mixtures of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. In the present invention, it is preferable to use a soft diamond.
  • the substrate is formed of at least one material selected from diamond, soft diamond, a carbon-based material, and carbide
  • it is not necessary to form a new surface layer on the substrate When the substrate is formed of another material, a surface treatment is performed to form a surface layer formed of at least one material selected from diamond, soft diamond, a carbon-based material, and carbide.
  • a soft diamond film was formed on a slide glass.
  • a substrate is preferably formed by ionization vapor deposition in a mixed gas containing 0 to 99% by volume of hydrogen gas and 100 to 1% by volume of methane gas remaining.
  • the thickness of the surface layer formed by the surface treatment is preferably 1 nm to 100 ⁇ m.
  • the surface treatment of the substrate may be performed by a known method, for example, a microwave plasma CVD (Chemical Vapor Deposit) method, an ECRCVD (Electric and yclotron Resonance to hemicai Vapor Deposit) method, an ICP (Inductive Coupled Plasma) method, a DC sputtering method.
  • a microwave plasma CVD Chemical Vapor Deposit
  • ECRCVD Electro and yclotron Resonance to hemicai Vapor Deposit
  • ICP Inductive Coupled Plasma
  • DC sputtering method a known method, for example, a microwave plasma CVD (Chemical Vapor Deposit) method, an ECRCVD (Electric and yclotron Resonance to hemicai Vapor Deposit) method, an ICP (Inductive Coupled Plasma) method, a DC sputtering method.
  • ECR Electro Cyclotron Resonance
  • EB Electrode
  • EB Electrode
  • resistance heating evaporation ionization evaporation
  • a surface layer may be formed by forming a laminate or a composite of the above-mentioned substrate material (for example, a composite of diamond and another substance (for example, a two-phase body)).
  • the shape and size of the substrate are not particularly limited. Examples of the shape include a flat plate, a thread, a sphere, a polygon, a powder, and the like. Has a width of about 0.1-100 mm, a length of about 0.1-100 mm, and a thickness of about 0.01-10 mm. Further, a single layer of Ti, Au, Pt, Nb, Cr, TiC, TiN or the like or a composite film thereof may be formed on the front surface or the back surface of the substrate as a reflective layer. The thickness of the reflective layer is preferably 10 nm or more, more preferably 100 nm or more, since it is necessary that the thickness of the reflective layer be uniform throughout.
  • the solid support of the present invention may be provided with an electrostatic layer for electrostatically attracting the polypeptide.
  • the electrostatic layer is not particularly limited as long as the polypeptide electrostatically attracts the polypeptide and improves the amount of immobilization of the polypeptide, and for example, a compound having a positive charge such as an amino group-containing compound. Can be formed.
  • the amino group-containing conjugate includes an unsubstituted amino group (-NH 2) or a group having 1 carbon atom.
  • R is a substituent monosubstituted with an alkyl group, such as, for example, ethylenediamine, hexamethylenediamine, n-propylamine, monomethylamine, dimethylamine, monoethylamine, Getylamine, arylamine, aminoazobenzene, amino alcohols (eg, ethanolamine), atalinol, aminobenzoic acid, aminoantraquinone, amino acids (glycine, alanine, norin, leucine, serine, threon, cysteine) Methionine, phenylalanine, tryptophan, tyrosine, proline, cystine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine), adiline, or a polymer thereof (for example, polyallylamine, polylysine) Copolymers; polyamines (polyvalent amines)
  • the amino group-containing compound is introduced into a film forming apparatus to contain an amino group.
  • a carbon-based film is formed.
  • the electrostatic layer is formed without being covalently bonded to the surface layer
  • the non-substituted or mono-substituted substrate is formed on the substrate in order to increase the affinity between the electrostatic layer and the surface layer, that is, the adhesion. It is preferable to introduce a functional group capable of covalently binding to the polypeptide after depositing the compound having the amino group and the carbon compound.
  • the carbon compound used here is not particularly limited as long as it can be supplied as a gas.
  • the conditions of ionization vapor deposition which is preferred by ionization vapor deposition, are preferably such that the operating pressure is in the range of 0.1 to 50 Pa and the accelerating voltage is in the range of 200 to 1000 V.
  • the surface of the substrate having the surface layer is chlorinated by irradiating ultraviolet rays in chlorine gas to chlorinate the surface.
  • a polyvalent amine such as polyallylamine, polylysine, 4,4,, 4 "-triaminotriphenylmethane, triamterene, etc. is reacted, and bonded to the substrate to form a terminal on the opposite side.
  • an electrostatic layer can be formed.
  • a reaction for introducing a functional group capable of covalently binding to a polypeptide is performed in a solution on a substrate provided with an electrostatic layer.
  • a functional group capable of covalently binding to a polypeptide after immersing the substrate in a solution containing the above-mentioned compound having an unsubstituted or monosubstituted amino group.
  • the solvent for the solution include water, N-methylpyrrolidone, and ethanol.
  • the thickness of the electrostatic layer is preferably lnm-500 ⁇ m.
  • the solid support of the present invention has a functional group capable of covalently binding to a polypeptide.
  • the functional group can be formed by chemically modifying the substrate surface.
  • Examples of the functional group include a carboxyl group, an active ester group, a haloformyl group, a hydroxyl group, a sulfate group, a cyano group, a nitro group, a thiol group, and an amino group.
  • the compound used for introducing a carboxyl group as a functional group includes, for example, a compound represented by the formula: X-Ri-COOH (where X is a halogen atom, R 1 is a divalent carbon atom having 11 to 12 carbon atoms) Represents a hydrogen group), for example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, odoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chloroacetic acid Benzoic acid; a dicarboxylic acid represented by the formula: HOOC-R 2 -COOH (wherein R 2 represents a single bond or a divalent hydrocarbon group having 11 to 12 carbon atoms), for example, oxalic acid, malonic acid, succinic acid Acid, maleic acid, fumaric acid, phthalic acid; polycarboxylic acid such as polyacrylic acid, polymethacrylic acid, trimelli
  • the carboxyl group introduced as described above can be combined with a dehydration condensing agent such as cyanamide ⁇ carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarboimide) and N-hydroxyl.
  • a dehydration condensing agent such as cyanamide ⁇ carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarboimide) and N-hydroxyl.
  • Active esterification can be performed with a compound such as succinimide.
  • Examples of the compound used to introduce a haloformyl group as a functional group include, for example, Formula: X- OC- R 6 - CO- X (wherein, X is a halogen atom, R 6 represents a divalent hydrocarbon group of a single bond or a C 1 one 12) Gino ⁇ of the dicarboxylic acid represented by Rides such as succinic chloride and malonic chloride.
  • the compound used for introducing a hydroxyl group as a functional group includes, for example, a compound represented by the formula:
  • HO-R 7 -COOH (wherein, R 7 represents a divalent hydrocarbon group having 1 to 12 carbon atoms) is exemplified by a hydroxy or phenolic acid.
  • Examples of the compound used for introducing an amino group as a functional group include amino acids.
  • a surface treatment layer preferably a soft diamond layer
  • a functional group capable of covalently bonding to the polypeptide in particular, a terminal lipoxyl group bonded to an end bonded via an amide bond.
  • a solid support having a hydrocarbon group having the following as a carrier it is preferable that the functional group is activated by N-hydroxysuccinimide or the like.
  • Such a solid support firmly immobilizes the polypeptide and can firmly bind not only a purified polypeptide but also a polypeptide contained in an unpurified sample such as a crude cell extract. In particular. As a result, it becomes possible to detect the interaction between the soluble receptor derivative and the polypeptide with high sensitivity.
  • the carrier on which the polypeptide prepared above is immobilized is subjected to blocking before reacting with the soluble receptor derivative.
  • Blocking can be carried out by immersing in a blocking agent commonly used in the art, for example, 0.1 M Tris hydrochloric acid buffer containing 2% BSA and shaking for about 1 hour. The immersion temperature at this time is room temperature. Then, wash with PBS containing 0.1% Tween20 and centrifuge dry. After the blocking treatment as described above, a solution containing a soluble receptor derivative is added. Then, the cells are incubated at a normal room temperature for 0.5 to 3 hours to react the extracellular domain of the soluble receptor derivative with the polypeptide on the carrier.
  • the carrier is washed.
  • the washing liquid those commonly used in the art can be used.
  • 2X SSC an aqueous solution containing 0.2% SDS (sodium dodecyl sulfate), a PBS containing 0.1% Tween 20 and the like can be used. it can.
  • the soluble receptor derivative remaining on the carrier is detected by binding to the polypeptide on the carrier.
  • the polypeptide immobilized on the spot to which the soluble receptor derivative was bound was found to bind to the extracellular domain of the soluble receptor derivative, that is, to interact with the receptor from which the derivative was derived. .
  • the interaction between the soluble receptor derivative and the polypeptide on the carrier can be detected by a method commonly used in the art.
  • the soluble receptor derivative can be labeled with a label commonly used in the art, and detected based on a signal derived from the label.
  • Such labels include radioisotopes, enzymes (eg, alkaline phosphatase, acid phosphatase, peroxidase, j8-galactosidase, glucose-6-phosphate dehydrogenase and luciferase), fluorescent dyes (eg, the fluorescein series, the rhodamine series, Examples include the eosin series, the NBD series, and the like, and specific examples thereof include Cy5, Cy3, IC5, IC3, fluorescein, tetramethylrhodamine, Texas Red, ataridine orange, and the like, and chemiluminescent molecules.
  • enzymes eg, alkaline phosphatase, acid phosphatase, peroxidase, j8-galactosidase, glucose-6-phosphate dehydrogenase and luciferase
  • fluorescent dyes eg, the fluorescein series, the rhodamine series
  • Examples include the e
  • the interaction can also be detected using a specific substance and a substance having affinity for the substance.
  • a soluble receptor derivative is piotined, the soluble receptor derivative is allowed to react with the polypeptide on the carrier, and then avidin labeled as described above is added thereto, and the avidin and piotin are bound.
  • the soluble receptor derivative bound to the polypeptide on the carrier can be detected.
  • Specific substances that can be used in such an embodiment Z Examples of combinations of substances having an affinity therefor include, for example, a biotin-binding protein Z such as avidin and streptavidin, a maltose-binding protein Z maltose, and a G protein Z guar.
  • polyhistidine peptide Z Metal ions such as nickel or cobalt, daltathione-s-transferase Z daltathione, DNA binding protein ZDNA, antibody Z antigen molecule (epitope), calmodulin Z calmodulin binding peptide, ATP binding protein ZATP, S Tradiol receptor protein z
  • Various receptor proteins such as estradiol And its ligands, as well as ketone groups Z hydrazide groups, diol groups Z hydrazide groups, azide groups Z alkyl groups, and psoralen Z nucleobases (nucleobases such as a pyrimidine ring or purine ring or analogs thereof).
  • biotin-binding protein z-biotin such as avidin and streptavidin, is most preferred.
  • the carrier after the interaction can be directly analyzed by laser desorption Z ionization time-of-flight mass spectrometry.
  • Matrix-assisted laser desorption (MALDI) is the preferred ionization method.
  • the present invention also relates to a method for diagnosing a specific disease by detecting an interaction between a polypeptide and a soluble receptor derivative.
  • the interaction between a polypeptide and a soluble receptor derivative is detected, the amount of a ligand for a specific membrane receptor contained in a sample derived from a patient is measured, and the membrane level is determined based on the measured value.
  • Diseases associated with the scepter and its ligands can be diagnosed. For example, biological samples derived from various parts of a patient are spotted on a carrier, the polypeptide contained in each sample is immobilized on the carrier, and the interaction with a membrane receptor is detected to detect the interaction with the membrane receptor.
  • a large amount of a ligand for a control membrane receptor is expressed, and the strength of the membrane receptor can be analyzed, whereby a simple and effective diagnosis can be performed.
  • a solid support having a functional group capable of covalently bonding to a polypeptide on a substrate and at least one surface layer selected from diamond, soft diamond, a carbon-based substance, and a carbide.
  • a polyvalent alcohol it is preferable to add to a biological sample for spotting.
  • the polypeptide can be immobilized without purifying the biological sample, and the interaction with the membrane receptor can be detected.
  • Examples of a combination of a membrane receptor or a ligand and a disease related thereto include ErbBl receptor which is highly expressed in cancer cells, and a ligand which binds to the cell to promote cell proliferation and cause an increase in lesions. Combinations with diseases are included. Since the ligand that binds to ErbBl is not limited to one type, even if a specific factor in a sample is detected, the true causative factor may be missed. However, by using a soluble receptor to detect factors in the sample, we can detect factors that increase lesions in patient samples. Evaluation and diagnosis.
  • autoimmune diseases It is known that this disease produces antibodies (autoantibodies) in the blood that recognize their own cell surface substances, and that these cells attack their own cells, causing chronic inflammation.
  • the autoantibody can be regarded as a ligand for the cell surface, and by using this method, the autoantibody in the patient's blood is spotted, and the autoantibody is recognized by reacting with the soluble receptor. It is possible to obtain information and make a diagnosis. Also, a peptide or compound that neutralizes an autoantibody can be obtained by screening a small molecule recognized by the autoantibody.
  • ErbB 1 extracellular domain (ErbB 1 ECD)-FLAG
  • PC012-EGFR-bearing cells were picked up with a platinum loop and streaked on LB agar medium (LB-Amp50 Agar) containing 50 ⁇ g / ml ampicillin for colony isolation.
  • LB-Amp50 Agar LB agar medium
  • the cells were inoculated into 5 ml of an LB medium (LB-Amp50) containing 50 ⁇ g / ml ampicillin, and cultured at 37 ° C with shaking at 280 / min.
  • the cultured Escherichia coli was centrifuged at 3,000 rpm for 10 minutes at room temperature, collected, and the plasmid was extracted with a QIAprep Spin Miniprep Kit (QIAGEN).
  • the extracellular domain (ECD) gene was amplified.
  • An Xhol site was created on the N-terminal side of ErbBl, and an Agel site was created upstream of the transmembrane domain of ErbBl.
  • the reaction mixture was precipitated with ethanol using ethatin mate (NIPPON GENE), and the precipitate was sterilized with 18 ⁇ l of sterilized UPW.
  • Adenine was tilled at the 3 'end of the previously amplified ErbBl ECD gene.
  • the reaction solution was subjected to agarose electrophoresis, and a 2 kbp band was cut out.
  • DNA was extracted using the Extraction System (LIFE TECHNOLOGIES). This was precipitated with ethanol, and the precipitate was dissolved in 101 sterile UPW.
  • the band of ErbBl extracellular domain (ErbBl ECD) was confirmed by agarose electrophoresis, and the molar ratio of ErbBl extracellular domain and pCR2.1 (TA cloning vector, Invitrogen) was mixed at a ratio of 100: 1.
  • T4 DNA ligase (NIPPON GENE) was added and the mixture was reacted in a 16 ° C water bath.
  • the entire amount of the ligation reaction solution is aseptically added to the Combinent Cell XL-1 Blue, placed on ice for 30 minutes, heat-shocked at 42 ° C for 1 minute, immediately placed on ice, and sufficiently cooled to bring the SOC medium to 400 mL. Transformation was performed by culturing the cells at a temperature of 37 ° C for 1 hour. The culture solution was spread on LB-Amp50 Agar along with 50 ⁇ l of 20 mg / ml X-Gal and 10 ⁇ l of lOOmM isopropyl thiogalatatoside (IPTG) and cultured overnight at 37 ° C. for blue-white selection.
  • IPTG isopropyl thiogalatatoside
  • Step 1 1.Sterile UPW 17.25 ⁇ 1, Taq DNA polymerase 0.25 / zl mixed, set in thermal cycler, 95 ° C for 10 minutes, 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° The reaction was performed under the conditions of 1 minute at C, 30 cycles, and 10 minutes at 72 ° C. The reaction solution was subjected to agarose gel electrophoresis, and a 700 bp band was observed. Three clones were inoculated into 5 ml of LB-Amp50, and cultured at 37 ° C with shaking at 37 ° C.
  • Plasmids were extracted using the QIAprep Spin Miniprep Kit, and the insertion of ErbBl ECD into the cloning vector was confirmed by digestion with XhoI and Agel and EcoRI. Furthermore, the entire nucleotide sequence of ErbBl ECD was confirmed using a DNA sequencer (The primers used were # 361 (SEQ ID NO: 7), # 547 (SEQ ID NO: 27), # 548 (SEQ ID NO: 8), M13 (-21) primer (sequence No. 14) (Applied Biosystems)).
  • the plasmid whose sequence was confirmed was digested with XhoI and Agel, and the reaction solution was subjected to agarose gel electrophoresis to cut out the ErbBl ECD (2 kbp) band.
  • DNA from excised gel using CONCERT TM Rapid Gel Extraction System was extracted and ethanol precipitated with ethatin mate, and the precipitate was dissolved in 101 sterile UPW.
  • pEGFP-Nl (pB0315) was cut with Xhol and Agel, and the restriction enzyme was inactivated at 70 ° C for 15 minutes.
  • the ErbBl ECD gene and pB0315 were mixed at a molar ratio of 100: 1, 1 ⁇ l of T4 DNA ligase (NEB) was added, and the mixture was allowed to react at ⁇ 16 ° C.
  • the whole amount of the reaction solution was introduced into XL-1 Blue for transformation, inoculated on an LB agar medium containing 50 g / ml kanamycin (LB-Kn50 Agar), and cultured at 37 ° C at ⁇ ° C. Five emerged colonies were picked, inoculated into 5 ml of 50 g / ml kanamycin-containing LB medium (LB-Kn50), and cultured with shaking.
  • Plasmids were extracted with the QIAprep Spin Miniprep Kit, respectively, cut with Xhol and Agel, and it was confirmed that ErbBl ECD was inserted into pB0315. After confirmation, one clone was cut with Agel and Notl to eliminate the EGFP sequence from PB0315. Agarose electrophoresis was performed to cut out a 6 kbp fragment. DNA was extracted from the cut gel with the CONCERT TM Rapid Gel Extraction System and dissolved in 10 ⁇ l of sterile UPW.
  • the FLAG-tag of the linker was 100 ⁇ , 2.5 ⁇ l each of synthetic oligonucleotides # 536 and # 537 (SEQ ID NOS: 15 and 16), 2 ⁇ l of 10 mM ATP, and 10 ⁇ L of 10X T4 polynucleotide kinase.
  • the previously prepared vector fragment excluding EGFP and FLAG-tag were mixed at a molar specific force of l: 100, 1 ⁇ l of T4 DNA ligase was added, and the mixture was allowed to react at 16 ° C. Was. All the reaction solutions were used for XL-1 Blue transformation, and the transformants were inoculated into LB_Kn50 Agar. After the culture, the colonies were picked and inoculated into 5 ml of LB-Kn50, and cultured at 37 ° C with shaking. Then, the plasmid was extracted with the QIAprep Spin Miniprep Kit, and insertion of FLAG was confirmed using restriction enzymes.
  • the clone in which the insertion was confirmed was inoculated into 2 ml of LB-Kn50 and cultured with shaking at 37 ° C for 7 hours.
  • the culture solution was transferred to another 250 ml LB-Kn50 in 1 ml, and cultured with shaking (150 rpm) in an orbital shaker. Cultures discarded 4 ° C with the centrifuge 5000 rpm, 7 minutes (HITACHI himac CR20) o supernatant was transferred to a 500ml high speed centrifuge tube and transferred to a 50ml high speed centrifuge tube by suspending the cell pellet in a small volume of Ringer's solution.
  • the precipitate was dried, and 3 ml of TE8.5 was dissolved in each centrifuge tube to dissolve the precipitate, and 3.3 g of cesium chloride was dissolved in the precipitate. These were transferred to two tubes for ultracentrifugation, and 50 ⁇ l of 20 mg / ml ethidium bromide was added to each tube, and the tube was pressed and stoppered. The tube was set on the rotor and ultracentrifuged at 62000 rpm for 6 hours at 20 ° C.
  • the tube was removed from the rotor, and a 21-gauge injection needle (Teremo) was inserted into the shoulder of the tube, and the band of the plasmid was extracted with an lml syringe (Tenoremo) fitted with an 18-gauge needle (Tenoremo). At that time, 495 light was applied to the tube to fluoresce the band.
  • the extracted liquid was transferred to a 1.5 ml tube, 400 ⁇ l of TE-saturated butanol was added, vortexed and centrifuged for 3 minutes, and the organic phase (upper layer) stained red was aspirated with a pipetman. This operation was repeated five times to remove ethidium bromide.
  • the solution from which this ethidium bromide has been removed is transferred to two 15-ml glass centrifuge tubes, mixed with 3 times the volume of sterilized UPW, mixed with 8 volumes of ethanol, mixed well, and placed on ice. Placed for minutes. Then, the mixture was centrifuged at 4 ° C and 13,000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was dissolved in a total of 1 ml of TE8.5. Transfer these to four 1.5 ml tubes, add 10 1 of each to 3M sodium acetate (pH 5.2), vortex, further add 520 ⁇ l of ethanol, vortex and vortex into a dry ice-ethanol bath. Immerse for 30 minutes and centrifuge for 10 minutes.
  • the precipitate was washed with ethanol, dried, and dissolved in a total of 400 ⁇ l of 10 mM Tris-HCl pH8.5 ImM EDTA (TE8.5).
  • the cells and the plasmid were stored at -80 ° C and -20 ° C, respectively, as pBO507.
  • the leverage reaction solution was added to each of 11 and 10X PFU buffer, and the 10 M forward primer # 534 (SEQ ID NO: 5) and the 10 M reverse primer # 535 (SEQ ID NO: 6) were added in 1 ⁇ l each for 2.5 ⁇ l.
  • reaction solution was dissolved in 18 ⁇ l of sterilized UPW using ethatin mate. To this, add 2.5 ⁇ l of 10X PCR buffer, 3 ⁇ l of 10 mM dATP, 1 ⁇ l of 2.5 mM dNTP, and 0.5 ⁇ l of Taq DNA polymerase, and react at 72 ° C for 1 hour. Adenine was tilled at the 3 'end.
  • primers # 361 SEQ ID NO: 7
  • # 549 SEQ ID NO: 9
  • # 550 SEQ ID NO: 10
  • # 563 SEQ ID NO: 11
  • # 564 SEQ ID NO: 13
  • M13 M13 (-21) primer
  • the DNA base sequence was confirmed using SEQ ID NO: 14).
  • the vector was cut with Agel and Bglll and subjected to agarose gel electrophoresis to cut out the ErbB4 ECD band. DNA was extracted from the gel using the CONCERT TM Rapid Gel Extraction System and dissolved in 10 ⁇ l of sterile UPW.
  • PB0315 was also cut with Agel and Bglll, and the reaction solution was left at 65 ° C for 15 minutes to inactivate the restriction enzyme. Then, the ErbB4 ECD gene and FLAG-tag were mixed at a molar specific force of 1: 100, 1 ⁇ l of T4 DNA ligase was added, and the mixture was allowed to react at ⁇ 16 ° C. Then, transfer the entire amount of the reaction solution to XL-1 Blue. The transformant used for transformation was inoculated into LB-Kn50Agar, and cultured at 37 ° C overnight.
  • the emerging colony was inoculated into 5 ml of LB-Kn50, cultured, extracted with a QIAprep Spin Miniprep Kit, cut with Agel and Bglll, and screened. Thereafter, the FLAG-tag was inserted in the same manner as in To1, and the plasmid was purified in large quantities by cesium salt density gradient ultracentrifugation.
  • CIAA phenol / cloform form isoamyl alcohol
  • aqueous layer upper layer, colorless and transparent
  • phenol / CIAA was added again, and the same operation was repeated twice.
  • 100 ⁇ l of CIAA was vortexed and centrifuged for 5 minutes to collect the water tank.
  • the mouse IgGl hinge region has 100 ⁇ synthetic oligonucleotides # 584 and # 585 (SEQ ID NOS: 18 and 19), 2.5 each, 10 mM 2 21 and 10 X T4 10 buffer 101 and sterile UPW 82 ⁇ l. 1 Put 1 ⁇ l of ⁇ 4 ⁇ into a 0.2 ml tube, set in a thermal cycler, react at 37 ° C for 1 hour, then 5 minutes at 95 ° C, 5 minutes at 60 ° C, 37 ° C For 30 minutes.
  • HEK293H Human embryonic kidney 293H (HEK293H) cells were cultured in 293 SFM II (GIBCO). The cells were collected and dispensed into two 1.5 mL tubes at 8 ⁇ 10 6 cells. This was centrifuged to remove the supernatant, and EP medium (10 mM glucose, 100 ⁇ M DTT / RPMI1640) was added and suspended in 800 ⁇ L portions. Then, 10 ⁇ g of plasmid DNA: ErbB1 ECD-hinge-FLAG expression vector (pB0547) or ErbB4 ECD-hinge-FLAG expression vector (pB0548) was added, and the mixture was transferred to a 4 mm gap-elect-portion cuvette (M ⁇ ). .
  • the plasmid was introduced at 0.26 kV-950 ⁇ F using Gene pulser ⁇ ( ⁇ -RAD). 10% FCS-DMEM was placed in a T75 cm 2 cell culture bottle, and the cells subjected to electoral poration were added. Incubate for 24 hours at 37 ° C, 5% CO
  • the culture solution is collected, centrifuged at 1200 rpm for 5 minutes at 4 ° C, the supernatant is removed, and the cells are removed in 2 mL lmg / mL G418, 100 ⁇ g / mL kanamycin I 293 SFMII (lmg / mL
  • G418-293 SFM II G418-293 SFM II
  • the number of viable cells was counted (ErbB1 ECD-hinge-FLAG: 8.2 ⁇ 10 5
  • ErbB4 ECD-hinge-FLAG 6.4 ⁇ 10 5 ).
  • 18 mL of lmg / mL G418-293 SFM II was placed in a T75 cm 2 bottle, and the cells were collected.
  • G418 resistant cells were selected by culturing for 17-21 days.
  • Biotin- (AC5) 2-Sulfo-OSu Sulfosuccinimidyl N- [ ⁇ '-(D-Biotul) -6-Aminohexanoyl] -6'-Aminohexanoate
  • Dojindo Dissolve to make a 3.3 mM solution.
  • the reaction was stopped by adding 50 L of 0.1 M glycine, the total volume of the reaction solution was adjusted to 2.5 mL with UPW, applied to a PD-10 column (Amersham pharmacia biotech) equilibrated with PBS, and eluted with 3 mL of PBS. Buffer exchanged.
  • Construct Myc-BTC-HA expression plasmid (pB0651), introduce it into E.coli BL21 (DE3) pLysS, and achieve OD660 of 0.3 in LB medium containing 50 ⁇ g / mL ampicillin and 10 ⁇ g / mL chloramuecol. Cultured until Thereafter, isopropylthiogalactoside (IPTG) was added to a final concentration of 0.4 mM, and the cells were further cultured for 3 hours. The culture was centrifuged to collect the cells, frozen at _80 ° C, and thawed at 37 ° C.
  • IPTG isopropylthiogalactoside
  • the cell pellet was suspended in 1/50 volume of 50 mM phosphate buffer (pH 7.4), suspended on ice for 1 hour, and lysed. The supernatant was recovered by centrifugation and used as a crude Myc-BTC-HA extract.
  • An electrostatic layer was formed by immersing a 25 mm (width) x 75 mm (length) xlmm (thickness) glass slide in a polyallylamine aqueous solution (0.1 g / l). Thereafter, polyacrylic acid as a polycarboxylic acid was condensed with the amino group of the electrostatic layer in the presence of 0.1 M 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide.
  • the solid support was immersed in 15 mL of 2% BSA, 0.1 M Tris-Cl (pH 7.4) / PBS to perform blocking at room temperature for 1 hour.
  • the solid support was washed three times with PBS containing 0.1% Tween-20 (PBST), centrifuged, and then covered with a cover glass over 30 L of 100 g / mL biotinylated ErbBl ECD-hinge-FLAG containing 1% BSA.
  • the reaction was performed at room temperature for 1 hour.
  • the plate was washed three times with PBST and centrifuged.
  • Figure 4 shows the results of analyzing the fluorescence intensity using GTMASS ANALYSIS. From the above results, it can be seen that spots with a larger amount of immobilized polypeptide interacting with ErbB have more ErbB extracellular domains bound thereto. As described above, it was shown that the interaction of the polypeptide with the membrane receptor can be detected by the method of the present invention.
  • an interaction between a receptor and a polypeptide can be detected quickly and easily, and screening of a ligand for a receptor and screening of a ligand mutant having a desired binding activity to a receptor become possible.

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Abstract

L'invention concerne un procédé pour détecter, de manière rapide et commode, une interaction entre un polypeptide et un récepteur. Ce procédé comprend les étapes qui consistent : à immobiliser le polypeptide sur un support dont la couche superficielle comporte un groupe fonctionnel et une substance carbonée ; à induire une réaction avec un dérivé récepteur qui comporte un domaine extracellulaire de récepteur membranaire sur une surface de cellule, mais qui ne comporte aucune région indispensable à la liaison membranaire, et ; à détecter toute interaction entre le polypeptide immobilisé et le dérivé récepteur.
PCT/JP2004/018347 2003-12-11 2004-12-09 Procede pour detecter une interaction entre un polypeptide et un recepteur, procede pour cribler un ligand ou un variant de ligand au moyen de ce procede de detection, et procede de diagnostic utilisant ledit procede de detection WO2005062045A1 (fr)

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JP2007282591A (ja) * 2006-04-19 2007-11-01 Yamaguchi Univ スタスミンチップ及びこれを用いたスタスミン結合タンパク質の検出方法
JP2008105973A (ja) * 2006-10-24 2008-05-08 Toyo Kohan Co Ltd ポリペプチド固定化担体の保存方法
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