JPWO2007083793A1 - Panning method using photoreactive group and kit used therefor - Google Patents

Abstract

A method for screening a protein or peptide that interacts with a ligand more reliably is provided. A photoreactive ligand and a protein-displayed phage or a peptide-displayed phage are mixed in a liquid phase, irradiated with an active light to the photoreactive group of the photoreactive ligand, and the photoreactive ligand has an immobilization function. Protein-displayed phage or peptide-displayed phage bound to the photoreactive ligand immobilized on the solid phase by washing the solid phase immobilized with the photoreactive ligand on the solid phase A method of screening for a protein or peptide that interacts with a photoreactive ligand, comprising recovering. [Selection] Figure 4

Description

Cross-reference of related applications

  This application claims the priority of Japanese Patent Application No. 2006-14314 filed on Jan. 23, 2006, the entire description of which is specifically incorporated herein by reference.

  The present invention relates to a panning method for efficiently isolating a display phage using a photoreactive group such as phenyldiazirine. Hereinafter, this method is referred to as a photopanning method. The present invention relates to a method for efficiently screening proteins or peptides that interact with a ligand using this photopanning method. Furthermore, the present invention relates to a kit used for this screening method.

  A technique for displaying a protein or peptide on the surface of a phage and screening a phage displaying the target protein or peptide was completed in 1985-1990. (G. P. Smith (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 228, 1315-1317. (Non-Patent Document 1), SE Cwirla, EA Peters, RW Barrett, WJ Dower (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73, 305-318, SF Parmley, GP Smith. (1990) Peptides on phage: a vast library of peptides for Proc. Natl. Acad. Sci. USA 87, 6378-6382 (Non-patent Document 3))

Displayed phages can survive under fairly powerful denaturing conditions, can be used as if dealing with protein molecules rather than living organisms, and the genes that express them can be easily isolated. Widely used in The panning method most often used as a screening method for the displayed phages is derived from “pan” which means that the gold dust is washed and separated in a beneficiation pan, and the target displayed phages are separated by affinity with the ligand. Thereafter, the phage population enriched in the target phage is propagated. By repeating this, the target display phage is gradually concentrated and can be isolated (see FIG. 2). Since the phage has a dramatic growth efficiency exceeding 10 ¹⁰ within a few hours, it can be panned several times in one day. In recent years, a panning method for phages displaying antibodies has been expected as a basic technique for searching for seeds of antibody medicine using this high-speed screening ability.

  The present inventors have so far developed an original high-speed photoaffinity method using a diazirine derivative as a photoreactive group. The photoaffinity method is a technique for irreversibly linking a specific ligand and a partner protein using a photoreaction. Diazirine has the advantage of favoring this irreversible binding compared to other photoreactive groups. (Y. Sadakane, Y. Hatanaka (2004) Multifunctional photoprobes for rapid protein identification. In F. Darvas, A. Guttman and G. Dorman eds., Chemical Genomics, Marcel Dekker Inc., New York. Pp199-214) Reference 4), Y. Hatanaka, Y. Sadakane (2002) Photoaffinity labeling in drug discovery and developments: Chemical gateway for entering proteomic frontier. Curr. Top. Med. Chem. 2, 271-288 (Non-patent Document 5), Hatanaka Yasumaru Structural Biological Organic Chemistry: Probing Protein Functional Structures with Photoaffinity Labels, Journal of Organic Synthesis, 56 (7), 581-590,1998 (Non-patent Document 6), M. Kaneda, Y. Sadakane, Y Hatanaka, (2003) A Novel Approach for Affinity-Based Screening of Target Specific Ligands: Application of Photoreactive D-Glyceraldehyde-3-phosphate Dehydrogenase. Bioconjugate Chem. 14, 849-852. (Non-Patent Document 7), M. Hashimoto , J. Yang, Y. Hatanaka, Y. Sadakane, K. Nakagomi, GD Holman (2002) Improvement in the properties of 3-phenyl-3-tr ifluoromethyldiazirine based photoreactive bis-glucose probes for GLUT4 following substitution on the phenyl ring. Chem. Pharm. Bull. 50, 1004-1006 (Non-patent Document 8), Hatanaka, Y., Kempin, U., Park, JJ. One- step synthesis of biotinyl photoprobes from unprotected carbohydrates. J. Org. Chem. 65, 5639-5643, 2000 (Non-Patent Document 9), Hatanaka, Y., Hashimoto, H., Kanaoka, Y. A rapid and efficient method for identifying Photoaffinity biotinylated sites within proteins. J. Am. Chem. Soc. 120, 453-454, 1998 (Non-patent Document 10), JP 2000-319262 (Patent Document 1)).

The present inventors conducted preliminary experiments in anticipation of technological innovation by combining the characteristics of photoreactive groups and display phage panning. A photoreactive group having a chemically selective functional group was immobilized on a solid phase, and a solid phase into which a sugar chain ligand or a peptide ligand was introduced was prepared by using this functional group. From this preliminary experimental result, it was revealed that the displayed phage can be covalently immobilized on the solid phase and that the phage can be recovered from the solid phase. However, since this method had a limit on the amount of photoreactive ligand that could be immobilized on the solid phase, the amount of immobilized ligand was limited to an amount that could screen thousands of display phages per 1 cm ² at a time. . For this reason, the shortage of the amount of phage capture, which is thought to be affected by the insufficient amount of ligand on the solid phase, becomes a problem, and it requires several hundred times the area to be used for actual panning. It was not established as a technology. (Y. Sadakane, H. Nakashima, J. -J. Park, K. Nakagomi, Y. Hatanaka (2002) EFFICIENT CLONING OF CARBOHYDRATE BINDING PROTEINS. Photomedicine and Photobiology 23, 83-84 (Non-Patent Document 11), Y. Sadakane, H. Nakashima, J.-J Park, Y. Hatanaka (2002) Preparation of solid photo-crosslinking matrix and application to photo-panning of displayed phages. Photomedicine and Photobiology 24, 85-86. )
G. P. Smith (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.Science, 228, 1315-1317. SE Cwirla, EA Peters, RW Barrett, WJ Dower (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73, 305-318 SF Parmley, GP Smith. (1990) Peptides on phage: a vast library of peptides for identifying ligands.Proc. Natl. Acad. Sci. USA 87, 6378-6382 Y. Sadakane, Y. Hatanaka (2004) Multifunctional photoprobes for rapid protein identification.In F. Darvas, A. Guttman and G. Dorman eds., Chemical Genomics, Marcel Dekker Inc., New York. Pp199-214 Y. Hatanaka, Y. Sadakane (2002) Photoaffinity labeling in drug discovery and developments: Chemical gateway for entering proteomic frontier. Curr. Top. Med. Chem. 2, 271-288 Yasumaru Hatanaka Structural Biological Organic Chemistry: Probing Functional Protein Structures with Photoaffinity Labels, Journal of Organic Synthesis, 56 (7), 581-590,1998 M. Kaneda, Y. Sadakane, Y. Hatanaka, (2003) A Novel Approach for Affinity-Based Screening of Target Specific Ligands: Application of Photoreactive D-Glyceraldehyde-3-phosphate Dehydrogenase. Bioconjugate Chem. 14, 849-852. M. Hashimoto, J. Yang, Y. Hatanaka, Y. Sadakane, K. Nakagomi, GD Holman (2002) Improvement in the properties of 3-phenyl-3-trifluoromethyldiazirine based photoreactive bis-glucose probes for GLUT4 following substitution on the phenyl ring. Chem. Pharm. Bull. 50, 1004-1006 Hatanaka, Y., Kempin, U., Park, JJ.One-step synthesis of biotinyl photoprobes from unprotected carbohydrates. J. Org. Chem. 65, 5639-5643, 2000 Hatanaka, Y., Hashimoto, H., Kanaoka, Y. A rapid and efficient method for identifying photoaffinity biotinylated sites within proteins.J. Am. Chem. Soc. 120, 453-454, 1998 Y. Sadakane, H. Nakashima, J. -J. ParkJ, K. Nakagomi, Y. Hatanaka (2002) EFFICIENT CLONING OF CARBOHYDRATE BINDING PROTEINS.Photomedicine and Photobiology 23, 83-84 Y. Sadakane, H. Nakashima, J.-J Park, Y. Hatanaka (2002) Preparation of solid photo-crosslinking matrix and application to photo-panning of displayed phages .. Photomedicine and Photobiology 24, 85-86. JP, 2000-319262, A All the above-mentioned nonpatent literatures 1-12 and patent documents 1 are used as an indication here especially.

  The panning method, which is a technique for isolating the target display phage, is epoch-making compared to the screening methods known so far, and is considered to be applicable in various fields. However, the range of use is actually limited. There are two main reasons for this. First, whether or not the target display phage can be isolated is determined by the affinity between the ligand and the displayed protein or peptide. In the actual panning method, it is known that if the affinity between the two is not very strong, panning operation with good reproducibility cannot be performed and the concentration efficiency is not stable. As an improvement measure against this, improvement of the display phage expression system is being promoted mainly, and contrivances such as increasing the number of expressed proteins have been made. However, it is not a general method. Second, the amplification of the phages performed after the panning operation causes a difference in growth between the phages. In particular, it is known that when a large molecule such as a protein is displayed on a phage, the difference in growth becomes remarkable, and a phage that is overwhelmingly wild-type becomes dominant. For these reasons, it has been pointed out for a long time that there is a problem that the target display phage goes somewhere after repeated panning operations several times, and the wild-type phage is finally isolated. .

In order to solve the above problems, it is necessary to develop a selection method that does not rely on the affinity between the ligand and the displayed phage, and to thoroughly eliminate phages that are close to wild strains that remain due to nonspecific binding. is there. As a means to realize this solution, the idea of panning using a photoreactive group was conceived. As a preliminary experiment, the present inventors have already clarified that the display phage can be immobilized by a photoreactive ligand arranged on a solid phase. However, this reason the method is that there is a limit to the amount of the photoreactive ligand that could be immobilized on a solid phase, the purpose of using the panning intended for standard phage libraries, the several hundred cm ² solid Because the phase area is necessary, it has not been established as a practical technique, but only suggests a methodological possibility.

  The present invention aims at realizing the above-mentioned solution, and is a method for screening a phage displaying a target protein or peptide from a phage library displaying the protein or peptide on the phage surface. A protein that interacts with a ligand by providing a practical method for efficiently and specifically isolating a target display phage from a phage library on a matrix through a sufficient amount of a photoreactive ligand. Alternatively, it is an object to provide a method for screening a peptide more reliably than in the conventional method.

The present inventors have completed the present invention by finding that a target display phage can be isolated efficiently and reliably by using a photoreactive ligand in a conventional panning method.

The present invention for solving the above problems is as follows.
[1] A photoreactive ligand having a photoreactive group and a protein-displayed phage or peptide-displayed phage are mixed in a liquid phase,
Irradiate an active light to the photoreactive group of the photoreactive ligand,
Immobilize the photoreactive ligand on the solid phase,
Washing the solid phase with immobilized photoreactive ligand,
A method for screening a protein or peptide that interacts with a photoreactive ligand, comprising recovering a protein-displayed phage or peptide-displayed phage bound to a photoreactive ligand immobilized on a solid phase.
[2] The method according to [1], wherein the photoreactive ligand is a photoreactive peptide, a photoreactive protein, a photoreactive nucleic acid, or a photoreactive sugar chain.
[3] The method according to [1] or [2], wherein the photoreactive group is a diazirine group.
[4] Immobilization of a photoreactive ligand to a solid phase is performed using a biotin-avidin or streptavidin system, a His tag-nickel system, a boron-hydroxamic acid chelate system, or an antigen-antibody system [1] to [ The method according to any one of [3].
[5] (1) The photoreactive ligand has biotin and the solid phase is coated with avidin or streptavidin. (2) The photoreactive ligand has a His tag and the solid phase is coated with nickel. (3) the photoreactive ligand has a hydroxamic acid chelate, the solid phase is coated with boron, and (4) the photoreactive ligand has an antigen, and the solid phase contains an antibody. The method according to [4], which is any one coated.
[6] The method according to any one of [1] to [5], wherein the solid phase is a magnetic bead or a substrate.
[7] The method according to [6], wherein the protein-displayed phage or peptide-displayed phage bound to the photoreactive ligand immobilized on the solid phase is recovered by recovering the magnetic beads.
[8] The method according to any one of [1] to [7], wherein the cleaning is performed using a surfactant.
[9] The method according to any one of [1] to [8], wherein the protein or peptide is identified by identifying the DNA of the recovered protein-displayed phage or peptide-displayed phage.
[10] Proliferating the recovered protein-displayed phage or peptide-displayed phage, and using the propagated protein-displayed phage or peptide-displayed phage, mixing in a liquid phase with the photoreactive ligand described in [1], At least one irradiation of active light, immobilization of photoreactive ligand on solid phase, washing of solid phase, and recovery of protein- or peptide-displayed phage bound to photoreactive ligand immobilized on solid phase The method according to any one of [1] to [8], which is performed.
[11] The method according to [10], wherein the protein or peptide is identified by identifying the DNA of the recovered protein-displayed phage or peptide-displayed phage.
[12] A kit used for screening a protein or peptide that interacts with a photoreactive ligand,
The kit comprising a photoreactive ligand having a photoreactive group and a solid phase on which the photoreactive ligand is immobilized.
[13] The kit according to [12], wherein the photoreactive ligand is a photoreactive peptide, a photoreactive protein, a photoreactive nucleic acid, or a photoreactive sugar chain.
[14] The kit according to [12] or [13], wherein the photoreactive group is a diazirine group.
[15] The photoreactive ligand is immobilized using a biotin-avidin or streptavidin system, His tag-nickel system, boron-hydroxamic acid chelate system, or antigen-antibody system. The kit according to any one of [12] to [14], wherein
[16] (1) The photoreactive ligand has biotin and the solid phase is coated with avidin or streptavidin. (2) The photoreactive ligand has a His tag and the solid phase is coated with nickel. (3) the photoreactive ligand has a hydroxamic acid chelate, the solid phase is coated with boron, and (4) the photoreactive ligand has an antigen, and the solid phase contains an antibody. The kit according to [15], which is either coated.
[17] The kit according to any one of [12] to [16], wherein the solid phase is a magnetic bead or a substrate.
[18] The kit according to any one of [12] to [17], further comprising a cleaning agent for cleaning the solid phase on which the photoreactive ligand is immobilized.
[19] The kit according to any one of [12] to [18], which is used in the method according to any one of [1] to [11].
[20] A kit for producing a photoreactive ligand having a photoreactive group, comprising a compound having a photoreactive group, a ligand labeling substance, and a photoreaction mounting solution.
[21] The kit according to [20], wherein the compound having a photoreactive group is a phenyldiazirine compound represented by the following general formula (C).
(In the above formula, R ₆ is a halogen atom or a sulfonic acid ester residue, R ₇ is an alkylthiosulfonyl group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms, and R ₈ is H or an alkyl group having 1 to 6 carbon atoms, and n is an integer of 1 to 6)
[22] The kit according to [20] or [21], wherein the ligand labeling substance is biotin, a histidine tag (His tag), a hydroxamic acid chelate or an antigen.
[23] A kit for validation comprising a protein-displayed phage dilution solution and a photoreactive ligand solution.

  According to the present invention, it is possible to provide a panning method having a concentration effect approximately 1000 times that of the conventional panning method. A photoreactive ligand can capture a display phage having an affinity for it with a covalent bond. This irreversible binding makes it possible to remove most of the display phages adsorbed non-specifically without missing the target display phages even after powerful washing. When the photoreactive group is a diazirine group, the photoreaction that captures the display phage occurs efficiently and extremely quickly with light near 360 nm. In addition, this photoreaction does not damage the display phage and does not affect the growth ability of the phage necessary for the panning method.

  Furthermore, since a powerful cleaning operation can be performed, an empirical feeling is not required as in the case of conventional panning cleaning. As a result, proteins or peptides that interact with the ligand can be screened more efficiently and reliably. Furthermore, it is possible to mechanize the isolation operation of the display phage. In particular, mechanization of the panning operation is economically meaningful as a method for isolating antibody-displayed phages that have been developed in recent years.

[Screening method]
The present invention is a method for screening proteins or peptides that interact with a photoreactive ligand, the method comprising:
(1) A photoreactive ligand having a photoreactive group and a protein-displayed phage or peptide-displayed phage are mixed in a liquid phase,
(2) irradiating the photoreactive ligand with actinic light to the photoreactive group,
(3) Immobilizing the photoreactive ligand on the solid phase,
(4) Wash the solid phase on which the photoreactive ligand is immobilized,
(5) collecting the protein-displayed phage or peptide-displayed phage bound to the photoreactive ligand immobilized on the solid phase.

[Photoreactive ligand]
In the present invention, the photoreactive ligand is a ligand for a protein or peptide displayed by a protein-displaying phage or peptide-displaying phage, and has a photoreactive group. Here, the ligand is, for example, a peptide or protein, and more specifically, a peptide having one cysteine residue in the original sequence, a peptide having one cysteine residue introduced therein, and an appropriate site. There are peptides into which photoreactive amino acids have been introduced. Furthermore, there are proteins containing cysteine residues that are not involved in SS binding, or proteins into which cysteine residues have been newly introduced by a genetic modification method or the like. If these properties are satisfied, a peptide or protein having an arbitrary sequence can be used as a ligand.

  A photoreactive group is a chemical reactive group that takes an intermediate that is highly reactive when irradiated with light of a specific wavelength, and the intermediate has a property of forming a covalent bond with the nearest molecule to stabilize it. Examples of the photoreactive group include a diazirine group, an azide group, and a carbonyl group. In the present invention, the photoreactive group is preferably a diazirine group.

  A ligand equipped with a photoreactive group is called a photoreactive ligand, and examples of the photoreactive ligand include a photoreactive peptide, a photoreactive protein, a photoreactive nucleic acid, and a photoreactive sugar chain.

[Method for producing photoreactive peptide ligand]
A photoreactive peptide ligand can be prepared by introducing a photoreactive group into a peptide that can be a ligand. The following two methods can be used to introduce a photoreactive group into a peptide that can be a ligand.

  One is a method for introducing a photoreactive group through a cysteine residue, and the other is a method for producing using a photoreactive amino acid. In a sequence containing a cysteine residue in the peptide ligand, a photoreactive group can be introduced through the sequence. Further, in the case of a ligand having a sequence not containing a cysteine residue, a photoreactive group can be bound by introducing a cysteine residue at an arbitrary position. If a photoreactive amino acid is used, a photoreactive peptide can be synthesized using a peptide synthesizer or the like.

  Hereinafter, the method for preparing a photoreactive peptide ligand will be further described with reference to a photoreactive calmodulin-binding peptide ligand as an example.

In this example, two types of photoreactive calmodulin-binding peptide ligands were produced using a calmodulin-binding peptide (17 residues) as a template.
A photoreactive peptide ligand was prepared based on the calmodulin-binding peptide of the following sequence 1. This peptide has been confirmed to recognize and specifically bind calmodulin protein. Two photoreactive peptides shown in Sequence 2 and Sequence 4 with different production methods were synthesized. One is a photoreactive peptide obtained by synthesizing a peptide in which the third tryptophan from the N-terminus is replaced with a photoreactive amino acid tmd (Phe) and biotinylating the N-terminus. (Sequence 2). The chemical structure is shown under Sequence 2. Using Fmoc-tmd (Phe) (compound 2) in which the Fmoc protecting group was added to the amino group of tmd (Phe) (compound 1), a photoreactive peptide was prepared with a peptide synthesizer. The other is to synthesize a peptide in which the third tryptophan from the N-terminus is replaced with cysteine (sequence 3), biotinylate the N-terminus, and then introduce the diazirine shown in compound 3 with a thiol bond. Sex peptide (sequence 4). The two types of photoreactive calmodulin-binding peptides synthesized were purified by HPLC and used.

[Production method of photoreactive protein ligand]
A photoreactive protein ligand can be produced by attaching a photoreactive group to calmodulin. For protein ligands, recombinant calmodulin protein (molecular weight of about 20,000) with Cys residues introduced is produced in E. coli, and a photoreactive group is attached to the produced protein. Examples of production of three types of photoreactive calmodulin protein ligands with different sites for introducing photoreactive groups are shown below.

  It was prepared by attaching diazirine thiosulfonate (compound 3), which specifically binds to a cysteine residue, to the cysteine residue introduced by genetic modification. Three types of photoreactive calmodulin proteins with different photoreactive group introduction sites were prepared. From the three-dimensional structure analysis result (PDB: ICDL) of the MLCK fragment peptide that is calmodulin and its binding peptide, it can be seen that the 26th, 75th, and 115th amino acids are in different positions with the MLCK fragment peptide as the center. Each amino acid was converted to cysteine and mounted with compound 3. Thereafter, each was biotinylated. The following (Scheme 1) shows the positions of the calmodulin protein and the 115th lysine. Calmodulin is a protein that originally does not contain cysteine residues. Calmodulin was prepared in Escherichia coli by modifying the gene, having 6 histidines at the N-terminus and mutating amino acids 26, 75 and 115 to cysteine (scheme 2). Six histidines are tags required for purification of the expressed protein. In the examples, evaluation experiments were performed using calmodulin-binding peptide-displayed phages and S-tag-displayed phages.

  Specifically, 0.5 mL of purified expressed protein (2 mg / ml), 0.015 mL of compound 3 (1 mol / L) and 0.5 mL of acetonitrile were mixed and left overnight at 4 ° C. to produce the protein shown in schematic diagram 3. . Excess reagent was removed by gel filtration. The purified photoreactive calmodulin and biotin OSu were allowed to stand at 4 ° C overnight under the condition of pH 9.0 to produce biotinylated photoreactive calmodulin. Excess reagent was removed by gel filtration to obtain biotinylated photoreactive calmodulin. (Scheme 4) How many biotins are attached to one calmodulin molecule is determined by determining the amount of biotin by a quantitative method using 2-hydroxyazobenzene-4'-carboxylic acid (HABA) and comparing it with the amount of calmodulin. be able to.

[Photoreactive nucleic acid]
The photoreactive nucleic acid ligand can be, for example, one obtained by attaching a photoreactive group to a nucleic acid in which the phosphate group of at least one nucleic acid such as DNA, RNA, or PNA having a photoreactive group has a thiophosphate group.

  The photoreactive nucleic acid ligand can be produced by reacting at least one nucleic acid phosphate group with a thiophosphate group and a phenyldiazirine compound having a reactive group with the thiol group of the thiophosphate group. Among DNA and RNA, those having a thiophosphate group are known and can be appropriately synthesized by known methods described in the following documents. (Zon, G .; Geiser, TG (1991) Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and future directions.Anticancer Drug Des. 6: 539-568. And Caruthers, MH; Beaton, G .; Wu, JV; Wiesler, W. (1992) Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs. Methods Enzymol. 211: 3-20. The entire description thereof is hereby specifically incorporated by reference.)

  The nucleic acid can be converted into a nucleic acid derivative equipped with a biotin tag by a known method in advance. (S. Agrawal; C. Christodoulou; MJ Gait (1986) Efficient methods for attaching non-radioactive labels to the 5 'ends of synthetic oligodeoxyribonucleotides Nucleic Acids Res. 14: 6227-6245. "Nonisotopic DNA probe techniques" (ed. LJ (See Kricka) Academic Press, New York, the entire description of which is specifically incorporated herein by reference.)

  The phenyldiazirine compound that is a photoreactive group can be, for example, a compound represented by the following general formula (C).

In the above formula, R ₆ is a halogen atom (eg, Cl, Br, I), or a sulfonic acid ester residue. Specifically, the sulfonic acid ester residue can be a methanesulfonyl group or a toluenesulfonyl group. R ₇ is an alkylthiosulfonyl group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms. Alkylthio alkylsulfonyl group, RSO ₂ - indicated, R is for example, be methyl, ethyl, propyl or butyl. The alkylthio group is represented by RS- and R can be, for example, methyl, ethyl, propyl or butyl. R ₈ can be H or an alkyl group having 1 to 6 carbon atoms, and the alkyl group can be, for example, methyl, ethyl, propyl, or butyl. n is an integer of 1 to 6, preferably an integer of 1 to 4, and more preferably 1.

  The above-mentioned phenyldiazirine compound having a thiol-reactive group and a nucleic acid derivative are mixed at a molar ratio of, for example, about 50: 1 to 100: 1. Furthermore, diisopropylethylamine in a molar ratio of 50 to 100 times with respect to the nucleic acid derivative is added to this mixture, and the reaction can be carried out in dimethyl sulfoxide at 37 ° C. However, N-methylmorpholine, triethylamine or the like can be used instead of diisopropylethylamine. As the reaction solvent, methanol, dimethylformamide or the like can be used instead of dimethyl sulfoxide. The reaction temperature can be in the range of, for example, 4 to 70 ° C., including 37 ° C.

  The concentration of the phenyldiazirine compound is suitably in the range of 0.1 to 0.5 mM, for example. The reaction time depends on the type of the phenyldiazirine compound, but can be, for example, about several tens of minutes to several hours. It is preferable to store in liquid nitrogen after the reaction so as not to cause excessive reaction. The product phenyldiazirine-added nucleic acid derivative can be purified by, for example, high performance liquid chromatography. In addition to high performance liquid chromatography, for example, it can be purified by electrophoresis or the like.

[Photoreactive sugar chain]
The photoreactive sugar chain ligand can be various monosaccharides or oligosaccharides having an aldehyde terminus. Examples of monosaccharides include a pyranose ring and a furanose ring, such as glucopyranose (glucose), glucofuranose, and epimers such as galactose and mannose. Examples of the oligosaccharide include polysaccharides such as Lewis X from disaccharides such as maltose, sucrose, and lactose. Any sugar having an aldehyde terminal can be used to produce a photoreactive sugar chain ligand, and is not limited to the above example.

  A photoreactive sugar chain ligand can be simultaneously attached to a photoreactive group and biotin at the aldehyde end of the sugar chain by the method shown in the following paper. (See Hatanaka, Y., Kempin, U., Park, JJ. One-step synthesis of biotinyl photoprobes from unprotected carbohydrates. J. Org. Chem. 65, 5639-5643, 2000 (non-patent document 9))

[Protein-displaying phage]
A protein-displaying phage is a phage that displays on the surface a protein that is expected or expected to interact with a ligand. The protein may be single (one type) or multiple types. For example, any display protein may be a population of phage displayed based on a cDNA library, which is referred to as a “library”.

  Any protein or peptide can be displayed on the surface of the phage. For example, “protein-displaying phage” includes a type displayed on the foot of the phage (M13 type) and a type displayed on the head (FIG. 1) (T7 type). Any “protein-displayed phage” can be used in the present invention. By using “protein-displaying phage”, a protein having affinity for a specific receptor protein can be searched. Similarly, a peptide having affinity for a specific ligand can be found by using a peptide-displayed phage.

  A method for preparing a protein- or peptide-displayed phage can be performed by a conventional method. Proteins and peptides to be presented can be produced based on cDNA. cDNA can be produced from mRNA prepared from an organ or the like. A phage population (library) capable of preparing cDNA from a specific organ or the like and displaying a protein or peptide can also be purchased as a commercial product. Moreover, an antibody can be mentioned as a protein displayed on a phage, and a phage displaying an antibody protein is also called a phage antibody. When screening phage antibodies, a library of phage antibodies can be used.

The photopanning method of the present invention will be described below.
First, the photopanning method of the present invention will be schematically described with reference to FIG.
1. Photoreactive ligands are used in the photopanning method (leftmost step).
2. When a display phage having affinity for a ligand is bound to the ligand by affinity and light irradiation is performed at that moment, the ligand and the display phage are covalently bound (second step from the left).
3. Under any washing conditions, phages linked by this irreversible bond (covalent bond) will not be removed. Intense washing removes non-specifically bound phage that are not captured covalently (such as phage that are not bound to a ligand) (third step from the left).
4. The captured display phage can be propagated in E. coli to make the first phage sample, and photopanning can be repeated. (The fourth step from the left).

  Further, the photopanning method of the present invention will be described in detail with reference to FIG. The photopanning method of the present invention mainly comprises (A) a photoreactive calmodulin-binding peptide and a photopanning method for isolating a calmodulin-displaying phage, and (B) a photoreactive calmodulin protein and a calmodulin-binding peptide. Although there is a photopanning method for isolating the displayed phage, FIG. 4 illustrates an example of the method using the photoreactive calmodulin-binding peptide (A).

[Mixing] (FIG. 4, schematic diagram 5)
In the method of the present invention, the photoreactive ligand and protein-displayed phage are mixed in a liquid phase. Mixing in the liquid phase can be performed as follows. That is, a solution that satisfies the condition that the ligand and the target protein can be physiologically bound is used. A photoreactive ligand and protein-displayed phage are added thereto, and the mixture is stirred and mixed. In order to promote the binding, after mixing, for example, it can be allowed to stand at 37 ° C. for about 10 to 60 minutes. Thereby, the selection efficiency can be improved.

  When a photoreactive ligand and a protein-displayed phage are mixed in a liquid phase, if the affinity between the photoreactive ligand and the protein of the protein-displayed phage exceeds a certain level, they interact to form a complex. Can do. A complex consists of weak bonds (interactions) such as hydrogen bonds and hydrophobic interactions that occur between proteins and ligands.

[Activation light irradiation] (FIG. 4, schematic diagram 6)
The complex formed in the above mixing step is irradiated with active light for the photoreactive group of the photoreactive ligand, whereby the photoreactive ligand and the protein-displayed phage are covalently bound. As a result, a complex formed by a weak bond (interaction) is formed by a strong bond called a covalent bond. Irradiation with active light can be performed as follows.

[Light irradiation conditions]
The wavelength of the active light varies depending on the photoreactive group used, but in the case of diazirine, light having a maximum wavelength of 360 nm is used. In the experiment, active light was irradiated using a commercially available UV-A lamp. This lamp is a lamp having a maximum wavelength of 360 nm. When the photoreactive group is other than diazirine, the wavelength of the active light to be irradiated can be appropriately selected according to the type of each photoreactive group.

The complex formed in the solution is irradiated with active light to form a covalent bond. It is thought that 360 nm light is hardly absorbed by the solution, and irradiation reaches each component without blocking the light. Light irradiation can be performed from both the upper and lower parts of the solution at an intensity of, for example, 30 W / m ² to promote the photoreaction. The intensity of light can be appropriately determined in consideration of the composition of the solution.

  Due to the photochemical nature of diazirine, covalent bonds can be formed in a relatively short irradiation time. As for the irradiation time, 1 minute is sufficient from the result of the evaluation experiment (1) in FIG. 6 of Experiment 1. However, in order to complete the covalent bond formation, for example, 2 minutes is appropriate. is there. However, it is not intended to be limited to 2 minutes. Under these conditions, irradiation with active light for 10 minutes did not change the viability of subsequent phages. Therefore, the irradiation time can be appropriately determined within a range of 10 minutes or less, for example.

  Next, the photoreactive ligand possessed by the covalently bound complex is immobilized on a solid phase. Immobilization can be performed using, for example, a biotin-avidin system, a His tag-nickel chelate system, a boron-hydroxamic acid chelate system, an antigen-antibody reaction system, or the like.

  In the case where a photoreactive ligand is immobilized in advance, free contact with the binding display phage is inhibited, and it is considered that panning efficiency is reduced. Therefore, in the present invention, for example, a method can be used in which a photoreactive ligand is biotinylated, for example, and immobilized with avidin using this tag. Thereby, the photoreactive ligand and the display phage can be freely contacted in the solution, and it is considered that the binding by the affinity with the specific display phage can be promoted.

  The solid phase can be, for example, a magnetic bead, a substrate, or the like. Furthermore, the solid phase has any function that can be used for the immobilization. For example, if the photoreactive ligand has biotin, the solid phase can be avidin or streptavidin coated. As other solid phases, when the photoreactive ligand has a His tag, the solid phase is coated with nickel, and when the photoreactive ligand has a hydroxamic acid chelate, the solid phase is coated with boron. When the photoreactive ligand has an antigen, the solid phase can be coated with an antibody.

[Properties of immobilized avidin] (FIG. 4, schematic diagram 7)
The complex can be recovered with immobilized avidin using, for example, biotin introduced into the photoreactive ligand as a tag. There are various types of immobilized avidin. For example, avidin immobilized on a polystyrene plate and avidin immobilized on a magnetic bead can be used. The same applies to immobilization by a system other than the biotin-avidin system.

[Plate immobilized avidin]
Since the plate-immobilized avidin is washed on the plate, the background is very small and stable recovery efficiency is exhibited. A background that is about 1/10 times that of magnetic beads can be obtained. (See Table 3 for the results of the evaluation experiment (5))

However, the amount of ligand that can be immobilized is limited, and may not be suitable for library screening. Plate- immobilized avidin is in the form of a 96-well avidin-immobilized plate, and the amount of avidin immobilized on the plate is small (60 pmol / well). There is also a tendency for the amount of reactive ligand to decrease. Since it is common to use an amount of avidin that is several times greater than the amount of biotin to be recovered, only 30 pmol of ligand can be used. When the solution volume is 200 μL, the ligand concentration is 0.15 μmol / L. Since Kd of calmodulin and calmodulin-binding protein is 10 ⁻⁷ to 10 ⁻⁸ , separation is possible at this concentration. Preferably, avidin magnetic beads are used in combination with a ligand whose binding protein Kd is less than 10 ⁻⁷ .

  Since a sufficient amount of photoreactive ligands cannot be used with plate-immobilized avidin, avidin magnetic beads can be used when screening from large numbers of phage, such as a library of display phages, or when using ligands with low affinity. preferable.

[Avidin magnetic beads]
As the avidin magnetic beads, magnetic beads having a low background and a large amount of avidin immobilized are optimal for the photopanning method of the present invention. For example, NEB Streptavidin Magnetic Beads (Streptavidin Magnetic Beads) manufacturer code # S1420S can be used. With avidin magnetic beads, the display phage solution can be completely removed, and the nonspecific binding display phage (background) is low. The background can be reduced to approximately 1,000 to 10,000.

In the conventional panning method in which thorough washing cannot be performed, even if the same magnetic beads are used, the number of phages is about 1000 times larger in the background (see Table 5 in Evaluation Experiment (7)). If the immobilized amount of avidin is 1000 pmol / mg and about 1 mg of magnetic beads are used, avidin is 1000 pmol and biotin can be recovered at about 500 pmol. When the solution volume is 200 μL, the ligand concentration is 2.5 μmol / L. As a result, even a ligand having a binding protein Kd of about 10 ⁻⁶ can be used. By realizing this concentration, it can be used for screening of a considerable number of ligand-receptor proteins.

  Since avidin magnetic beads are easy to operate, the operation time is shortened and reproducibility is improved, and mechanization is possible. Furthermore, it is preferable to reduce nonspecific adsorption with magnetic beads by adding 10% BSA.

(FIG. 4, schematic diagram 7) By using magnetic beads for immobilized avidin, low background and stable recovery were realized. Because of the large amount of immobilization, even a display phage with low affinity (Kd = 10 ⁻⁶ ) can be recovered.

  Next, the solid phase on which the photoreactive ligand is immobilized is washed. This washing is for separating the protein-displayed phage that is not covalently bound to the photoreactive ligand from the covalently bound complex. A surfactant is preferably used for cleaning. The washing method and conditions are such that a large amount of washing solution is added to the solid phase and stirred for several minutes, and then the solution is discarded. This operation can usually be repeated about 5 times.

[Cleaning conditions and recovery of display phage] (FIG. 4, schematic diagrams 8 to 10)
[Determination of cleaning conditions] (FIG. 4, schematic diagram 8)
The washing conditions for isolating the display phage covalently bound to the photoreactive ligand can be appropriately determined in consideration of the properties of the display phage, the washing efficiency of the surfactant used for washing, and the like.

  Since some of the displayed phages are weak against alkalis and acids, it is appropriate to use a buffer and adjust the pH to 8.8, for example. This pH is the pH at which the Tris buffer can exhibit the maximum buffering capacity. However, other buffer solutions can be used.

  Furthermore, it is preferable to eliminate the ionic bonds by increasing the salt concentration. Therefore, for example, sodium chloride of about 0.2 to 1 mol / L can be used. As will be described later, when a high concentration of sodium chloride and a surfactant SDS are allowed to coexist, precipitation may occur. Therefore, it may be preferable to use a sodium chloride concentration of about 0.25 mol / L.

  In addition, since calcium ions are essential for binding between calmodulin and its binding peptide, it is appropriate to use a chelating agent such as EDTA in order to eliminate it.

  As the surfactant, for example, SDS, Triton-X100, Tween 20 and the like can be used, and can be appropriately determined in consideration of the cleaning efficiency by the surfactant. According to the experiments by the present inventors, it was found that SDS is most suitable for removing non-specific phages. The concentration of SDS can be in the range of 0.1 to 1%, and is preferably 0.5% in consideration of the effects on recovery and amplification by bringing it into the subsequent steps.

  The use of SDS has the advantage that the magnetic beads immobilizing avidin are not adsorbed to the container and the cleaning solution can be removed more easily. When a surfactant is not used, a small amount of magnetic beads are usually sucked up when a cleaning solution is taken by adsorption onto the container.

  Finally, it is preferable to use, for example, 50 mM Tris-HCl (pH 8.8), 0.25 M NaCl, 50 mM EDTA, 0.5% SDS as the washing solution. However, the intention is not limited to this cleaning solution.

  Displayed phages that are not covalently bound can be reduced to several thousand by washing this solution three times. In order to perform washing more reliably, in the experiment, washing is performed 4 to 7, preferably 5 to 6 times with a washing solution containing an SDS solution. In addition, in order to suppress the influence of SDS on the next step, it is preferable to finally wash once with a cleaning solution not containing SDS. However, the number of washings can be appropriately determined according to the screening system.

  After washing, the protein-displayed phage bound to the photoreactive ligand immobilized on the solid phase is recovered. Thereby, a protein that interacts with a photoreactive ligand, particularly a protein having a specific interaction can be screened. A protein can be identified by identifying the DNA of the recovered protein-displayed phage.

  However, in the present invention, it is preferable to proliferate the collected protein-displayed phage and repeat the process shown in FIG. 3 at least once using the proliferated protein-displayed phage. More preferably, the process shown in FIG. 3 is repeated 2 to 10 times.

  The recovered protein-displayed phage is propagated and mixed again with the photoreactive ligand in the liquid phase, irradiation with active light, immobilization of the photoreactive ligand on the solid phase, washing of the solid phase, and By recovering the protein-displayed phage bound to the immobilized photoreactive ligand, a protein having a specific interaction can be recovered with higher selectivity.

  The recovered protein-displayed phage can be propagated, for example, as follows. The recovered phage solution is infected with an E. coli strain serving as an appropriate host. This is grown in a solution such as LB medium and the host is lysed to grow phages.

[Recovery and amplification of phage] (FIG. 4, schematic diagram 9)
When T7 phage is used as a display phage, the protein or peptide to be displayed is expressed in the head, so that even when a covalent bond is formed with the ligand, the foot necessary for infection is intact.

  Therefore, it is appropriate to collect and amplify the display phage captured by covalent bonding in a state immobilized on magnetic beads. After thorough washing, the display phages captured in the magnetic beads (scheme 8) are diluted every 10-fold, placed in 0.5-1 mL E. coli solution, infected with mixing for 5-10 minutes, and top agar Mix and spread on plate. It is appropriate that the infection time does not exceed 15 minutes from the viewpoint of accurately measuring the number of phages.

  When using a T13 phage that expresses a protein or peptide in the foot, it is necessary to separate the phage from the ligand by treating with a protease such as trypsin in the state shown in FIG. 8, but this method is a general method. It can be implemented.

  A protein can be identified by identifying the finally recovered DNA of the protein-displayed phage.

[Preparation of phage solution] (Fig. 4, schematic diagram 10)
All the remaining display phage captured by the magnetic beads is put into an E. coli solution, and infection and amplification are performed according to a conventional method. Using the amplified phage solution, the photopanning is repeated from the schematic diagram 5 again. In the method of the present invention, photopanning can be performed twice a day, for example.

[kit]
The present invention includes the following three kits.
(A) Kit for preparing a photoreactive ligand having a photoreactive group (b) Panning kit (c) Validation kit

(A) Kit for preparing a photoreactive ligand having a photoreactive group The kit for producing a photoreactive ligand having a photoreactive group of the present invention comprises a compound having a photoreactive group, a ligand labeling substance, and a photoreaction mounting solution. including.

  The compound having a photoreactive group can be, for example, a phenyldiazirine compound represented by the general formula (C), and the phenyldiazirine compound represented by the general formula (C) is shown in the screening method of the present invention. Compound. The ligand labeling substance can also be, for example, biotin, a histidine tag (His tag), a hydroxamic acid chelate or an antigen. The photoreactive mounting solution is appropriately selected depending on the compound having a photoreactive group and a substance that becomes a ligand.

A procedure for preparing a photoreactive peptide and a protein by using a kit for preparing a photoreactive ligand having a photoreactive group is exemplified.
(1) A biotinylated peptide containing a cysteine residue or a protein containing a free cysteine residue that is not SS-bonded is prepared.
(2) The diazirine thiosulfonate of the kit is dissolved in the photoreaction mounting solution attached to the kit and mixed with the peptide or protein.
(3) Leave at room temperature or 37 ° C for several hours in the case of peptides and about 10 hours in the case of proteins.
(4) The peptide is purified by high performance liquid chromatography (HPLC), and the protein is purified by gel filtration. In the case of peptide ligands, use of an HPLC apparatus is suitable, but photoreactive ligands can also be purified by monomeric biotin.
(5) The photoreactive protein is loaded with biotin using the protein biotinylation reagent of the kit.
(6) The photoreactive biotinylated protein is purified by gel filtration. Although purification by gel filtration is suitable, it can also be purified by monomeric biotin.

(B) Panning kit The panning kit of the present invention is a kit used for screening a protein or peptide that interacts with a photoreactive ligand. This kit includes a photoreactive ligand having a photoreactive group and a solid phase on which the photoreactive ligand is immobilized.

  The photoreactive ligand contained in the kit of the present invention is the same as the photoreactive ligand described in the screening method of the present invention. The photoreactive ligand can be, for example, a photoreactive peptide, a photoreactive protein, a photoreactive nucleic acid, or a photoreactive sugar chain. The photoreactive group possessed by the photoreactive ligand can be, for example, a diazirine group.

  The solid phase for immobilizing the photoreactive ligand is also the same as the solid phase for immobilizing the photoreactive ligand described in the screening method of the present invention. Such a solid phase can be, for example, a magnetic bead or a substrate. In addition, the solid phase on which the photoreactive ligand is immobilized may be, for example, biotin-avidin or streptavidin system, His tag-nickel system, boron-hydroxamic acid chelate system, or antigen-antibody system. It can be fixed.

  More specifically, the combination of the photoreactive ligand and the solid phase is (1) the photoreactive ligand has biotin, the solid phase is coated with avidin or streptavidin, and (2) photoreactive The ligand has a His tag, the solid phase is coated with nickel, (3) the photoreactive ligand has a hydroxamic acid chelate, the solid phase is coated with boron, and (4) light The reactive ligand can have an antigen and the solid phase can be either coated with an antibody.

  The kit of the present invention may further include a cleaning agent for cleaning the solid phase on which the photoreactive ligand is immobilized. The cleaning agent can be, for example, an aqueous solution containing a surfactant, and examples of the surfactant can be those described in the screening method of the present invention. The cleaning agent may further contain a chelating agent such as EDTA.

  The panning kit of the present invention can be used for the screening method of the present invention.

More specifically, the above panning kit is:
(b1) Solid phase
(b2) Serum-containing albumin aqueous solution
(b3) Cleaning solution
(b4) A phage recovery solution can be included.

  As described above, the solid phase is coated with adivine, streptavidin, nickel, boron or an antibody, and the solid phase carrier can be a magnetic bead or a substrate. A preferred solid phase is a magnetic bead coated with streptavidin. A buffer solution can be appropriately used as the phage recovery solution.

A procedure for performing photopanning using a protein-displayed phage using the panning kit of the present invention will be exemplified.
The phage used here is, for example, T7 phage, and a T7 phage library and E. coli for infection are prepared separately.
(1) Mix and leave in a solution that satisfies the conditions for interaction of photoreactive biotinylated ligand and T7 phage library.
(2) Using an ultraviolet lamp (intensity 30 W / m ² ), irradiate active light for 1 to 10 minutes.
(3) Add 10% serum-containing albumin aqueous solution of the kit to a final concentration of 1%, and mix the amount having streptavidin at a molar ratio of at least twice the amount of ligand using the kit's streptavidin magnetic beads.
(4) Shake at room temperature for 10-30 minutes.
(5) Add about 1mL of the kit washing solution and shake for 5 minutes.
(6) Collect the magnetic beads with a magnetic stand.
(7) Add about 1 mL of the washing solution to the collected magnetic beads, shake for 5 minutes, and collect on a magnetic stand.
(8) Repeat the above operation 4-6 times.
(9) Add about 1mL of the phage collection solution of the kit to the collected magnetic beads, shake for 5 minutes, and collect on a magnetic stand.
(10) A suitable amount of the collected magnetic beads are mixed with the infecting E. coli and shaken at 37 ° C. for 3 to 5 hours until the infecting E. coli is lysed.
(11) A suitable amount of the amplified phage solution and the photoreactive biotinylated ligand are mixed, and this operation (from the second procedure) is repeated 2 to 5 times.

(C) Validation Kit The validation kit of the present invention includes a protein-displayed phage dilution solution and a photoreactive ligand solution. For example, when the protein is calmodulin, the protein-displayed phage dilution solution may be a solution in which one calmodulin-displayed phage is mixed in 100,000 wild phages. The photoreactive ligand solution can be, for example, a photoreactive biotinylated calmodulin-binding peptide solution. The validation procedure using the validation kit of the present invention was carried out according to the panning procedure described above, using a diluted solution of calmodulin-presenting T7 phage instead of the library phage, and photoreactive biotinylation as a photoreactive biotinylation ligand. Perform using calmodulin-binding peptide solution.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Preparation of photoreactive peptide ligands Two photoreactive peptide ligands were prepared based on the calmodulin-binding peptide of sequence 1 that has been confirmed to recognize and specifically bind calmodulin protein.

[Production method using photoreactive amino acid (sequence 2)]
A peptide was synthesized in which the third tryptophan from the N-terminus of sequence 1 (LKWKKLLKLLKKLLKLG) (SEQ ID NO: 1) was replaced with tmd (Phe). The synthesis was performed by the Fmoc-solid phase synthesis method using a peptide synthesizer. Thereafter, a biotinylated peptide was synthesized by reacting the N-terminal amino group with biotin succinimide in a state where the synthesized peptide was in a solid phase. The synthetic peptide was dissociated from the solid phase with a strong acid, and the target biotinylated photoreactive peptide was purified by HPLC. Further, Fmoc-tmd (Phe) necessary for this synthesis was synthesized as follows.

Hatanaka, Y., Kempin, U., Park, JJ. One-step synthesis of biotinyl photoprobes from unprotected carbohydrates. J. Org. Chem. 65, 5639-5643, 2000 (Non-Patent Document 9) 4- [3- (Trifluoromethyl) -3H-diazirin-3-yl] benzaldehyde (12) was synthesized.
Thereafter, the following method L-4 ′-[3- (Trifluoromethyl) -3H-diazirin-3-yl] phenylalanine (15) was synthesized.
Furthermore, Fmoc-tmd (Phe) was synthesized by converting the amino group into Fmoc.

4- [3- (Trifluoromethyl) -3H-diazirin-3-yl] benzyl alcohol (13)
Compound (12) (3.3 g, 15.4 mmol) was dissolved in EtOH 7 mL, and NaBH ₄ (0.626 g, 16.5 mmol) suspended in EtOH 7 mL was added with stirring at 0 ° C. After the addition was completed, the mixture was returned to room temperature and stirred for 4 hours. To this, 1 M HCl was slowly added on ice to decompose excess reagent, followed by extraction with Ether, and the Ether layer was dried over magnesium sulfate. After removing the desiccant by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (Hexane: Ether = 1: 1) to obtain a pale yellow oily substance (13).

Yield 3.21 g (96%)
¹ H-NMR (CDCl ₃ )
σ: 7.39 (d, 2H, J = 8.5 Hz), 7.19 (d, 2H, J = 8.5 Hz), 4.71 (s, 2H), 1.87 (bs, 1H)

3- [4- (Bromomethyl) phenyl] -3- (trifluoromethyl) -3H-diazirine (14)
Compound (13) (344.0 mg, 1.59 mmol) was dissolved in 2 mL of CH ₂ Cl ₂ and CBr ₄ (658.2 mg, 1.985 mmol) was added. Cooled to _{0 ℃ Ph 3 P (474 mg} , 1.807 mmol) was added slowly. After returning to room temperature and stirring for 1 hour, the residue was purified by silica gel column chromatography (Hexane) to obtain a colorless oily substance (14).

Yield 401.8mg (90%)
¹ H-NMR (CDCl ₃ )
σ: 7.42 (d, 2H, J = 8.5 Hz), 7.17 (d, 2H, J = 8.5 Hz), 4.46 (s, 2H).

L-4 '-[3- (Trifluoromethyl) -3H-diazirin-3-yl] phenylalanine (15)
tert-Butylglycinate benzophenone imine (1.136 g, 3.839 mmol) and the asymmetric catalyst O (9) -Allyl-N-9-anthracenylmethylcinchonidium bromide ^[9] (0.2127 g, 0.351 mmol, 0.1 eq) in CH ₂ Cl ₂ 9 Dissolved in mL. Compound (14) (1.419 g, 5.085 mmol, 1.5 eq.) Was added, and the mixture was stirred at room temperature under an argon gas atmosphere. While cooling the reaction mixture to -78 ° C, 2-tert-Butylimino-2-diethylamino-1,3-dimethylperhirdo-1,3,2-diazaphosphorine (1.41 g, 5.139 mmol, Aldrich) was added dropwise in a few seconds, The reaction was carried out at -78 ° C for 7 hours. After returning to room temperature and distillation under reduced pressure, the residue was diluted with Ether and washed twice with distilled water and once with saturated saline. The oil layer was dried with magnesium sulfate. After removing the desiccant by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (Hexane: EtOAc = 7: 1) to obtain a yellow oily substance.

Yield 1.89 g (95%)
¹ H-NMR (CDCl ₃ )
σ: 7.57 (d, 2H, J = 6.9 Hz), 7.39-7.27 (m, 6H), 7.06 (q, 4H, J = 8.4 Hz)
6.59 (d, 2H, J = 6.9 Hz), 4.09 (q, 1H, J = 8.6, 4.6 Hz), 3.26-3.11 (m, 2H)
1.44 (s, 9H).

The obtained yellow oily substance (75 mg, 0.152 mmol) was dissolved in 1 mL of TFA at 0 ° C. and stirred at room temperature for 2 hours. After confirming the completion of the reaction with (TLC, Hexane: AcOEt = 7: 1), TFA was distilled off under reduced pressure, the residue was diluted with CH ₂ Cl ₂ and extracted three times with water. The aqueous layer was lyophilized to give a white solid (15).

Yield (as TFA salt) 53.4 mg (91%)
¹ H-NMR (CD ₃ OD)
σ: 7.46 (d, 2H, J = 7.9 Hz), 7.27 (d, 2H, J = 7.9 Hz), 3.84 (dd, 1H, J = 8.2, 4.5 Hz), 3.38 (m, 1H), 3.10 (d , 1H, J = 4.5, 8.2 Hz).
Optical purity (ee)

Thereafter, L-4 ′-[3- (Trifluoromethyl) -3H-diazirin-3-yl] phenylalanine (15) was induced into the Fmoc body. And the optical purity was confirmed by HPLC. After dissolving an appropriate amount in MeOH, eluting with a chiral column (Sumichiral OA-3300 2.5μm 4.6 mmφ × 25 cm) at 0.01 M Ammonium Acetate / MeOH at a flow rate of 1 mL / min, and by UV absorption derived from 360 nm diazirine. Detected.
ee = 98%
[a] _D + 22.4 ° (c = 0.52, EtOH)

[Production method using diazirine thiophosphate (sequence 4)]
A peptide in which the third tryptophan from the N-terminus of sequence 1 (LKWKKLLKLLKKLLKLG) (SEQ ID NO: 1) was replaced with cysteine was synthesized (sequence 3). The synthesis was performed by the Fmoc-solid phase synthesis method using a peptide synthesizer. Thereafter, a biotinylated peptide was synthesized by reacting the N-terminal amino group with biotin succinimide in a state where the synthesized peptide was in a solid phase. The synthetic peptide was dissociated from the solid phase with a strong acid, and the target biotinylated peptide was purified by HPLC. The diazirine shown in compound 3 and the purified peptide were mixed in a solution containing 30% acetonitrile to thiol bond the cysteine residue in the peptide with compound 3. The target biotinylated photoreactive peptide was purified by HPLC (sequence 4). Compound 3 required for this synthesis is M. Kaneda, Y. Sadakane, Y. Hatanaka, (2003) A Novel Approach for Affinity-Based Screening of Target Specific Ligands: Application of Photoreactive D-Glyceraldehyde-3-phosphate Dehydrogenase. Bioconjugate Chem. 14, 849-852 (Non-patent Document 7). Specifically, it is shown in the above synthesis method of compound 14.

Example 2
Preparation of biotinylated photoreactive calmodulin A calmodulin protein with one cysteine residue added was prepared by gene modification. Specifically, from a human brain cDNA library, by a PCR method, a CaM2 primer (5′-CTTAGGAGAATGCCTAACAGATG-3) (SEQ ID NO: 2) and a CaM3 primer (5′-CTGCAGGTCTTCATTTTGCAGTC-3 ′) (SEQ ID NO: 3) are used. Then, a partial gene of the calmodulin protein was amplified, and the calmodulin gene was amplified with the CaM1 primer (5′-GGATCCCCATGGCTGATCAGCTGACCGAAG-3 ′) (SEQ ID NO: 4) using this fragment as a new primer. By this megaprimer method, a gene for producing calmodulin in which the 115th lysine of calmodulin protein was modified to cysteine was obtained. This amplified fragment was inserted into a pQE32 vector (Qiagen) and transformed into E. coli. E. coli containing this gene was isolated and cultured in large quantities on LB medium. Thereafter, Escherichia coli was disrupted, and the calmodulin protein was purified with an HIS tag purification column. Calmodulins in which amino acids 26 and 75 were replaced with Cys residues were also produced in the same manner.

  0.5 mL of the purified expressed protein (2 mg / ml), 0.015 mL of compound 3 (1 mol / L) and 0.5 mL of acetonitrile were mixed and left overnight at 4 ° C. to produce the protein shown in FIG. Compound 3 required in this method was synthesized by the method described in [Production method using diazirine thiophosphate (sequence 4)].

  Excess reagent was removed by gel filtration. The purified photoreactive calmodulin and biotin OSu were allowed to stand at 4 ° C overnight under the condition of pH 9.0 to produce biotinylated photoreactive calmodulin. Excess reagent was removed by gel filtration to obtain biotinylated photoreactive calmodulin. (Schematic diagram 4)

Whether it was a biotinylated photoreactive calmodulin was identified by the following method.
A sample purified by electrophoresis was separated, transferred to a membrane, and then confirmed by Western blotting using an avidin-HRP reagent.

Example 3
[Experiment to evaluate the fabricated photoreactive ligand]
[Evaluation Experiment (1)]
As evaluation experiment 1, it was examined whether the photoreactive calmodulin-binding peptide (sequence 2) has the ability to recognize a calmodulin protein and form a complex bound by a covalent bond by light irradiation.

The photoreactive calmodulin-binding peptide (sequence 2) and calmodulin were mixed at a ratio of 1: 1 and allowed to stand at 37 ° C. for 10 minutes in the presence of calcium. Thereafter, ultraviolet light (peak wavelength: 360 nm) having an intensity of 30 W / m ² was irradiated for 1 minute to induce a photoreaction. FIG. 5 shows the sample analyzed by HPLC. The upper part is a sample irradiated with ultraviolet rays, and the lower part is a sample not irradiated with ultraviolet rays. From the results of mass spectrometry, the peak indicated by the arrow in FIG. 5 indicates that intact CaM: original calmodulin protein, cross-linked CaM: complex of calmodulin and photoreactive calmodulin-binding peptide, DA-CBP : Photoreactive calmodulin-binding peptide. The mass increase of 2410 in MALDI-TOF MASS is exactly the same as that of calmodulin-binding peptide.

  It can be seen from the result of “FIG. 5 No light irradiation” that the complex bound only by affinity is dissociated under the HPLC analysis conditions. In addition, the result of “FIG. 5 light irradiation 60 seconds” shows that the calmodulin protein and its binding peptide form a complex by covalent bond. FIG. 6 shows the result of quantifying the complex peak in “FIG. 5 light irradiation 60 seconds” at various irradiation times of ultraviolet rays and measuring the efficiency of complex formation. It became clear that the maximum efficiency was obtained by ultraviolet irradiation for 1 minute. About 3% of the initial calmodulin protein amount formed a complex.

  As in Evaluation Experiment 1, a complex of calmodulin protein and its binding protein was prepared and analyzed by SDS-PAGE. The electrophoresis in the upper part of FIG. 7 shows the result of preparing a sample by changing the irradiation time of ultraviolet rays. The method is described briefly on the right side of FIG. Since the high heat treatment is performed in the presence of the reducing agent, the complex other than the complex bound by the covalent bond is dissociated. In addition, since biotin attached to the photoreactive calmodulin peptide was detected with an avidinylated HRP enzyme, it cannot be detected with a low molecular weight binding peptide and calmodulin alone without biotin. It has been found that calcium is necessary for calmodulin protein complex formation, and the complex could not be detected in the presence of EGTA. The electrophoresis in the lower part of FIG. 7 shows the results of an inhibition experiment with a calmodulin antagonist. Complex formation is inhibited at 100 μmol / L TFP. The concentration dependence of this inhibition is in good agreement with that of calmodulin function inhibition.

[Summary]
From the evaluation experiment (1), it was revealed that the produced photoreactive calmodulin-binding peptide specifically recognizes calmodulin and forms a complex linked by covalent bond.

[Evaluation Experiment (2)]
As evaluation experiment 2, it was examined whether the photoreactive calmodulin protein (schematic diagram 3) has the ability to recognize a calmodulin-binding peptide and form a covalently bonded complex by light irradiation.

Photoreactive calmodulin protein (schematic diagram 3) and biotinylated calmodulin-binding peptide were mixed at a ratio of 1: 1 and allowed to stand at 37 ° C. for 10 minutes. Thereafter, ultraviolet light (peak wavelength: 360 nm) having an intensity of 30 W / m ² was irradiated for 1 minute to induce a photoreaction. The sample was analyzed by SDS-PAGE as shown in FIG. The presence or absence of ultraviolet rays and the presence or absence of calcium are shown in the lower part of FIG. Since the high heat treatment is performed, the complex other than the complex linked by the covalent bond is dissociated. In addition, since the biotin attached to the calmodulin peptide was detected by an avidinized HRP enzyme, the binding peptide itself having a small molecular weight cannot be detected. Although CBB staining shows almost the same amount of calmodulin under all conditions, chemiluminescence is detected only under conditions where both UV and calcium are present.

[Summary]
Evaluation experiment (2) revealed that the photoreactive calmodulin protein (schematic diagram 3) has the ability to recognize a calmodulin-binding peptide and form a complex.

Example 4
[Standard method of photopanning]
The procedure of the photopanning method constructed using the photoreactive calmodulin-binding peptide is shown below.
(1) A 200 μL solution composed of a display phage solution (10 ⁸ to 10 ⁹ ), 50 mM Tris / HCl (pH 7.4), and 1 mM CaCl ₂ is prepared.
(2) Add a photoreactive calmodulin-binding peptide (specifically, sequence 2 or sequence 4) to a final concentration of 10 μM and mix well.
(3) Leave at 37 ° C. for 10 minutes. (To promote binding by affinity between the display phage and ligand peptide)
(4) Transfer to a 96-well plate with a flat bottom made of polystyrene. At this time, dispense so that it does not exceed 100 μL per well.
(5) Irradiate with light for 2 minutes using an irradiation device with UV-A lamps with an intensity of 30 W / m ² arranged above and below.
(6) Transfer the solution from the 96-well plate to the microtube.
(7) Add 200 μL of 10% bovine serum albumin (BSA) and shake for 1 minute. (To reduce non-specific adsorption with magnetic beads)
(8) Add 100 to 250 μL of avidin-immobilized magnetic beads (NEB) and shake for 2 minutes. (This amount of magnetic beads can adsorb about 250 to 1000 pmol of biotin.)
(9) Centrifuge at 15000 rpm (about 20000 g) for 5 seconds, place on a dedicated magnetic stand (NEB) and gently remove the supernatant.
(10) Add 1 mL of washing solution (50 mM Tris / HCl (pH 8.8), 0.25 M NaCl, 50 mM EDTA, 0.5% SDS) and shake vigorously for 5 minutes.
(11) Centrifuge at 15000 rpm (about 20000 g) for 5 seconds, place on a dedicated magnetic stand (NEB), and gently remove the supernatant.
(12) Repeat steps (10) and (11) a total of 5 times.
(13) Add 1 mL of a solution containing 50 mM Tris / HCl (pH 8.8), 0.25 M NaCl, and 50 mM EDTA, and shake vigorously for 5 minutes.
(14) Centrifuge at 15000 rpm (about 20000 g) for 5 seconds, place on a dedicated magnetic stand (NEB) and gently remove the supernatant.
(15) Suspend the magnetic beads (precipitate) with 200 to 500 μL of SM buffer (0.1 M NaCl, 8 mM MgCl ₂ , 50 mM Tris / HCl (pH 7.5), 0.01% gelatin).
(16) Prepare a 10-fold dilution series suspension, take 50 μL of it, mix with 500 μL of E. coli solution (stock name: BLT5403, growth of OD600 = 1), and leave it for 5 minutes.
(17) Mix with 3mL of top agar (LB medium (tripton 10g, yeast extract 5g, NaCl 5g dissolved in 1L water) containing 0.7% agar) kept at 50 ℃. Spread on a plate (1.5% agar in LB medium and hardened in the plate).
(18) Let stand at 37 ° C. for about 2 hours and measure the number of plaques.
(19) The remaining suspension of (15) is mixed with 10 mL of E. coli solution and cultured for about 3 hours while shaking at 37 ° C. When the liquid is clear, remove E. coli by centrifugation and collect the supernatant.
Using this phage solution, the procedure from (1) is repeated.

[Evaluation experiment of photopanning method]
Evaluation Experiment (3) [Types of Photoreactive Peptide Ligand and Panning Efficiency]
Photopanning was performed using the photoreactive calmodulin-binding peptides of Sequence 2 and Sequence 4, and the selection efficiency of calmodulin-displayed phages was compared. (Table 1) Calmodulin-displayed phages (10 ⁸ ) and S-tag display control phages (10 ⁸ ) were selected using 100 pmol of photoreactive ligand according to the photopanning standard method described above. The number of phage trapped on the magnetic beads was measured. The experimental results are evaluated by how many times the number of calmodulin-displayed phage is captured relative to the number of control phages.

[Summary]
Comparing the number of calmodulin-displayed phages and control phages captured by the magnetic beads, it can be seen that the photopanning using the sequence 2 is 1000 times more efficient and the sequence 4 is 2000 times more efficient. Both have sufficient selection efficiency.

Evaluation Experiment (4) [Amount and Efficiency of Photoreactive Peptide Ligand]
The photoreactive calmodulin-binding peptide of sequence 2 was used, and the selection efficiency was compared by changing the amount of ligand. (Table 2) The amount of avidin magnetic beads used is 100 μL and has the ability to adsorb 250 pmol of biotin.

[Summary]
Sorting efficiency is poor in the 1000 pmol experiment using more ligand than the magnetic beads can adsorb. When using lower ligand amounts, the efficiency is not significantly affected.

Evaluation Experiment (5) [Comparison of Avidin Magnetic Beads and Avidin Plate]
Magnetic beads and plates were compared as immobilized avidin for recovering display phage. (Table 3)
The plate used was a 96-well avidin-immobilized plate (Pierce).

[Summary]
When comparing the number of calmodulin-displayed phages and the number of control phages, there was a difference of 1000 times for magnetic beads and 1800 times for avidin plates.

Evaluation experiment (6) [Efficiency determination of photopanning method]
Calmodulin-displaying phage and control phage were mixed in various proportions as shown in the table below. About 10 ⁹ phages were used in each experiment. Photopanning was performed using the photoreactive ligand (10 pmol) of sequence 2 and 100 μL of magnetic beads. The number of phage trapped on the magnetic beads was measured. Furthermore, plaque hybridization was performed using the calmodulin gene as a probe. Calmodulin-displayed phage plaques were regarded as positive plaques and compared with the total number of plaques, and the ratio was calculated. (Table 4)

[Summary]
The number of phage captured by the magnetic beads decreases as the proportion of calmodulin-displayed phage decreases.
Even when calmodulin phages were present at a ratio of 1 in 1,500, most of the phages captured by the magnetic beads were calmodulin-displayed phages.
Taking the example of 1: 15,000 (0.0066%) as an example, the number of calmodulin-displayed phages that were present in 1 in 15,000 increased to 333 (84%) in 400 by photopanning. . Since 0.0066% increased to 84%, it was 84 ÷ 0.0066 = 12727, which means that it was concentrated about 12700 times. Similarly, it was concentrated 13500 times at 1: 150,000 and 15000 times at 1: 1,500,000.
By this photopanning method, it was possible to concentrate about 13000 times in one operation. (The outline is explained in FIG. 9)

Evaluation Experiment (7) [Comparison between Photopanning Method and Conventional Panning Method (1)]
In order to compare the efficiency with the conventional panning method, a calmodulin-binding peptide in which the N-terminus of sequence 1 was biotinylated was prepared. This peptide (10 μmol / L) was used to determine how much calmodulin-displayed phage and control phage were captured by magnetic beads (100 μL) according to the standard method of photopanning (but without light irradiation). (Table 5) Washing was performed with a cleaning solution prepared by adding 0.05% or 0.5% surfactant Tween-20 to TBS (50 mM Tris / HCl (pH 7.4), 0.25 M NaCl) containing 1 mM CaCl ₂ . . In order to see the reproducibility, the same condition experiment ((1) and (2)) was performed twice.

[Summary]
The number of calmodulin-displayed phages captured by the magnetic beads does not change much, regardless of whether the conventional method or the photopanning method is used. However, when looking at the number of control phages, the conventional panning method captures about 1000 times more than the photopanning method. The difference in background is that the photopanning method can select target phages more efficiently than the conventional method. Comparing the experiments of (1) and (2), it can be seen that the conventional panning method has poor reproducibility.

Evaluation experiment (8) [Comparison between photopanning method and conventional panning method (2)]
Calmodulin-displaying phage and control phage were mixed in various proportions as shown in the table below. About 10 ⁸ phage were used in each experiment. A calmodulin-binding peptide (10 pmol) obtained by biotinylating the N-terminus of sequence 1 and a photoreactive ligand (10 pmol) of sequence 2 were used. Magnetic beads were performed at 25 μL.

The number of phage captured on the magnetic beads was measured for each ligand.
Each phage was appropriately diluted to allow 100 to 400 phage plaques to appear on one plate, and 5 mL of SM buffer was added thereon and shaken for 2 hours. PCR was performed on 2 μL of this solution using primers designed to amplify the calmodulin gene, and the presence or absence of calmodulin-displayed phages was confirmed in each dilution series. (Table 6 and FIG. 10)

[Summary]
The number of phages captured by the magnetic beads did not change significantly in the conventional method, but decreased in the photopanning method as the proportion of calmodulin-displayed phages decreased. The photopanning method has such a result because the number of background phages is reduced. From the results of PCR of the calmodulin gene, it can be seen that there is at least one calmodulin-displayed phage out of 300 when the conventional method is selected from (3) 1: 1,000 mixed phages. On the other hand, in the photopanning method, even when selecting from (6) 1: 1,000,000 mixed phages, it can be seen that at least 400 calmodulin-displayed phages are present. From the above PCR results, it has been clarified that the photopanning method has a selection efficiency at least 1000 times that of the conventional method. (The outline is explained in FIG. 11)

Example 5
[Library screening experiment]
[Method of experiment]
An experiment was conducted to isolate calmodulin-displayed phages from a cDNA-displayed phage library by the photopanning method and the conventional panning method. (See FIG. 12) Human brain cDNA expression library and human liver cDNA expression library were used. The human brain cDNA library is said to contain many calmodulin genes. Both purchased from Novagen, started the screening from approximately 10 ⁹ display phage. A photoreactive calmodulin-binding peptide of sequence 2 or a peptide obtained by biotinylating the N-terminus of sequence 1 was used as a ligand. The final concentration was 10 μmol / L in all cases. In the photopanning method, light irradiation was performed for 2 minutes. 50 μL of avidin magnetic beads were used. It has 12.5 times the amount of biotin-binding ability of the above biotinylated ligand. In the conventional method, TBS-0.05% Tw-Ca ²⁺ used in the evaluation experiment (7) was washed five times as a washing solution. A part of the phage solution after panning was appropriately diluted, and the number of phages captured on the magnetic beads was measured (FIG. 12, phage number measurement (2)).

  A plate formed at that time was used to produce a membrane, and plaque hybridization was performed using the calmodulin gene fragment as a probe, and the number of calmodulin-displayed phages was measured (FIG. 12, plaque hybrid). Furthermore, a small amount of SM buffer was put on the same plate, and a phage solution was prepared from the plate. Based on this solution, PCR was performed using primers that specifically amplify calmodulin to confirm the presence or absence of calmodulin-displayed phages (FIG. 12, PCR (2)). Amplified. After amplification, the number of phages was measured (FIG. 12, phage number measurement (1)), and the presence or absence of calmodulin-displayed phages was confirmed by PCR using a part (FIG. 12, PCR (1)).

[results of the experiment]
[Evaluation experiment (9) Photopanning method (human brain library) with or without light irradiation]
Calmodulin-displayed phages were screened from a human brain cDNA library. Using the ligand of sequence 2, the results were compared with and without light irradiation.

  The phages selected at the stage of Photo B (1) -2 (with light) were individually confirmed by the PCR method that amplifies the calmodulin gene (FIG. 13). The lane of M is a marker. The phage solution selected from Photo B (1) -1 (with light) to Photo B (1) -3 (with light) was appropriately diluted and cultured so that about 200 to 300 plaques appeared. A nitrocellulose membrane was placed there, and after appropriate operations, plaque hybridization was performed using the calmodulin gene as a probe (FIG. 14). The black spots are the phages displaying calmodulin (referred to as positive clones).

  To confirm reproducibility, the same experiment was performed (Table 8). The selected phage solution was diluted to form plaques, and the calmodulin gene was amplified by PCR using phages obtained from one plaque as a template (FIG. 15). Ten pieces were confirmed in each cycle.

  The result of amplifying the calmodulin gene by PCR using the phage solution after growth as a template is shown (FIG. 16). The selected phage solution was appropriately diluted, cultured so that about several hundred plaques appeared, and SM buffer was added to prepare a phage solution. The result of amplifying the calmodulin gene by PCR using this phage solution as a template is shown (FIG. 17). The numerical value under each electrophoresis indicates the approximate number of plaques that appeared on the used plate.

[Summary]
The number of pannings (Table 7) of Photo B (1) -3 (with light) is increased by a factor of 100 compared to Photo B (1) -1,2 (with light). This indicates that the phage solution has been homogenized to calmodulin-displayed phage, i.e., the rate has approached 100%.
The plaque hybridization results in Table 7 were determined from the results in FIG. In the first photopanning, 0.5%, 60% in the second, and 99% in the third, calmodulin-displayed phages are consistent with the results of the rapid increase in the number of phages described above.

  PCR that amplified the calmodulin gene using individual phage as a template (Fig. 13), 43 of the phages selected by Photo B (1) -2 were positive, and 43/60, or about 70%, displayed calmodulin. It turns out that it is a phage. This ratio almost coincides with the result obtained by plaque hybridization. In an experiment conducted using the amplified phage as a template (FIG. 16: Photo B (1) and (2)), amplification of the calmodulin gene can be observed by the second photopanning (with light). However, it was not amplified (without light). At each panning stage, the experiment (Fig. 17: Photo B (1) and (2)) was conducted to examine the presence of calmodulin-displayed phages in hundreds of phages. It turns out that it is obtained. In the case of (without light), calmodulin-displayed phages were not seen even after 3 pannings.

From the above results, it can be seen that light irradiation is necessary for the concentration of calmodulin-displayed phages. It becomes clear that the covalent bond formed by the photoreaction increases the efficiency of panning.
Further, from the experiment of Table 8 and FIG. 15, when isolating a calmodulin-displayed phage by the photopanning method, 0/10 (0%) at the first time, 7/10 (70%) at the second time, and 9 at the third time. / 10 (90%), almost the same as the result in the previous Table 7.
It can be said that the reproducibility is excellent.

[Evaluation experiment (10) Conventional method (human brain library)]
Attempts were made to isolate calmodulin-displayed phages by conventional methods. In order to confirm the reproducibility, the same experiment was performed twice.
The ligand used was a biotinylated N-terminus of sequence 1.

[Summary]
The number of phage after panning changes about 10 times in each cycle, but there is no reproducibility. From the results of plaque hybridization in Table 10, it can be seen that calmodulin-displayed phages are not concentrated. In an experiment conducted using the amplified phage as a template (FIG. 16: Conventional B (5) and (6)), the calmodulin gene is not amplified. In the experiment (Fig. 17: conventional B (5) and (6)) in which the presence of calmodulin-displayed phages in hundreds of phages was examined at each panning stage, Although increased, it disappeared at the fifth time and there is no reproducibility. As long as this system is used, the conventional panning method cannot concentrate calmodulin-displayed phages.

[Evaluation experiment (11) Photopanning method (human liver library)]
The photopanning method was evaluated using a library made from human liver cDNA, which is said to have a low calmodulin expression level. The experimental method is the same as the evaluation experiment (8).

[Summary]
From the results of plaque hybridization shown in Table 11, it can be seen that calmodulin-displayed phages can be enriched from liver cDNA to a rate of 21% by the third photopanning and 92% by the fourth. As a result of PCR performed in FIG. 18, this agrees well with 10% at the second time, 20% at the third time, and 90% at the fourth time. The abrupt increase in the number of phages in Table 11 occurs at the fourth time, and the occupation ratio of calmodulin-displayed phages is in good agreement. In an experiment conducted using the amplified phage as a template (FIG. 16: Photo L (4)), the calmodulin gene is visible from the third photopanning. In each of the panning stages, the calmodulin gene can be seen from the third photopanning in an experiment (Fig. 17: Photo L (4)) in which it was examined whether there existed calmodulin-displayed phages in hundreds of phages. The photopanning method was able to concentrate calmodulin-displayed phages even from a library with a small amount of expression, and almost 100%. The above results are summarized in FIG.

[Photopanning method using photoreactive calmodulin protein as a ligand]
[Evaluation experiment (12) Effect of the site where photoreactive groups are attached]
The efficiency of the photopanning method was compared between three photoreactive calmodulin proteins with different photoreactive group introduction sites (Table 12). From the three-dimensional structure analysis result (PDB: ICDL) of the MLCK fragment peptide that is calmodulin and its binding peptide, it can be seen that the 26th, 75th, and 115th amino acids are located at different positions centering on the MLCK fragment peptide. Each amino acid was converted to cysteine and mounted with compound 3. Thereafter, each was biotinylated. An evaluation experiment was performed using phage displaying calmodulin-binding peptide and phage displaying S tag. Regarding the method, the standard method described above was followed. The amount of photoreactive calmodulin ligand used was 30 pmol, and the amount of magnetic beads used was 100 μL.

[Summary]
Changing the site to which the photoreactive group was attached did not significantly affect the selection efficiency.

[Evaluation Experiment (13) Comparison between Photopanning Method and Conventional Method (1)]
Panning was performed between the calmodulin-binding peptide-displaying phage and the control phage, and the amount of phage captured by the magnetic beads was compared. The selection efficiency of the photopanning method and the conventional method was compared. As the photoreactive ligand, biotinylated 115-photoreactive calmodulin was used, and in the conventional method, biotinylated calmodulin protein was used. The conventional method was performed in the same manner as the experiment with peptide ligand (evaluation experiment (7)).

  The photopanning method can capture 100 times the amount of calmodulin-binding peptide-displayed phage as control phage. On the other hand, the conventional method was about 4 times (Table 13).

[Summary]
Even when a protein is used as a ligand, the photopanning method can select target phages more efficiently than the conventional method, as in the case of using a peptide ligand.

[Evaluation experiment (14) Comparison between photopanning method and conventional method (2)]
Calmodulin-binding peptide-displaying phage and control phage were mixed at various ratios as shown in Table 14 below. In each experiment, the total number of phages was adjusted to about 10 ⁸ . In the photopanning method, biotinylated photoreactive calmodulin protein was used as a ligand, and in the conventional method, biotinylated calmodulin protein was used as a ligand. The number of phage trapped in magnetic beads was measured (Table 14)

  In the conventional method, the number of phage captured by the magnetic beads does not change even when the proportion of binding phage decreases, but the number of captured phage decreases as the proportion decreases in the photopanning method. It was.

  The selected phage solution is diluted appropriately and cultured so that several hundred plaques appear. SM buffer is added to prepare a phage solution. Using this phage solution as a template, the gene for calmodulin-binding peptide is obtained by PCR. Amplified (Figure 20). The numbers on each electrophoresis correspond to the samples (1) to (4) shown in Table 14. Moreover, the lower numerical value shows the number of the plaques which appeared on the used plate. Only the coding region of the calmodulin-binding peptide shortens the gene to be amplified, making it difficult to detect by electrophoresis. Therefore, primers were prepared in the region encoding the peptide and the neighboring phage genome region, and PCR was performed. .

In the photopanning method, calmodulin-binding peptide-displayed phages were detected at all dilutions from 1: 100 to 1: 100,000, whereas in the conventional method, detection was not possible at a dilution of 1: 10,000.
The detection limit by PCR in the photopanning method is unknown and the number of phages on the plate varies, so it can not be determined accurately, but the photopanning method is at least 100 times more efficient than the conventional method It seems to be.

  The number of calmodulin-binding peptide-displayed phages in the phage solution captured from the diluted sample by the photopanning method was determined by plaque hybridization and is shown in FIG. The numbers at the top of the hybrid photos correspond to (1) to (4) in Table 14. In the lower row, the number of positive plaques / total number of plaques and the concentration ratio in parentheses are shown. Ten of the 680 calmodulin-binding peptide-displayed phages were recovered from the 1: 100,000 diluted phage solution. Therefore, the concentration efficiency is estimated to be 1470 times.

[Summary]
It became clear that the photopanning method had a concentration efficiency more than 100 times that of the conventional method, and it succeeded in the concentration of 1470 times in one photopanning.

[Evaluation Experiment (15) Library Screening Experiment]
Screening was performed with the aim of isolating proteins that bind to calmodulin from human brain and liver cDNA libraries. In the photopanning method, biotinylated photoreactive calmodulin protein was used, and in the conventional method biotinylated calmodulin protein was used at 30 pmol as a ligand, and screening was performed. Library was from about 10 ^{to nine,} including the screening. From the second time onward, the measurement was performed at 1/20 of the “concentration after growth” in the table corresponding to each time.
100 μL of avidin magnetic beads were used. In the conventional method, the cleaning conditions used in the evaluation experiment (7) were performed. The human brain library screened by the photopanning method is shown in Table 15, Table 17, and Table 18. Table 16 shows a screen of a human brain library by a conventional method. Table 19 shows a human liver library screened by photopanning.

[Summary]
In the photopanning method, the number of phages after panning increases as the cycle increases.

[Evaluation Experiment (16) Sequence of Isolated Phage Gene]
Cycles indicated by Godic in each table, Photo B (7) -4, Photo B (7) -5, Conventional method (-) B (8) -5, Photo B (9) -4, Photo B (10 ) -4, Photo L (11) -4, Photo L (11) -5 were isolated on the plate. The total number of isolated phages was 230. Using these as templates, the insert portion was amplified by PCR (example: FIG. 22). Among them, DNA of 400 bp or more was selected and the PCR fragment was recovered. A total of 228 fragments were recovered. For the recovered 228 fragments, the DNA sequence was read from the 5 'end.
Table 20 summarizes a plurality of recovered phages or phages having genes reported as calmodulin-binding proteins.

  Nos. 1, 2, 10, 11, 18 in the above table have already been reported as calmodulin-binding proteins. As a result of sequencing, it was found that the phage expressed the region including the calmodulin binding site in each protein in frame.

For No3, 7, 8, 9, 12, 13, 14, 15, 16, 17 in the table above, the Calmodulin Target Database (http://calcium.uhnres.utoronto.ca/ctdb/flash.htm )) Contains a sequence that has been evaluated as “8” or more by calmodulin binding analysis. This database was reported in Yap KL, Kim J, Truong K, Sherman M, Yuan T, Ikura M. J Struct Funct Genomics. 2000; 1 (1): 8-14. And has the ability to bind calmodulin. Can be evaluated.
FIG. 23 shows the analysis result of No3 AC092953 in the same database. The part below the sequence with a number of 8 or more is considered to bind to calmodulin.
In No3, 7, 8, 9, 12, 13, 14, 15, 16, 17, it is considered that the peptide does not exist in nature. That is, those that are read in the opposite strand, those that are frame-shifted, those that are reported not to be proteins.

  Regarding No. 4 and 6, calmodulin-binding ability has not been confirmed in the database. However, for these two phages, calmodulin binding activity is shown in a simple photopanning method (Table 21).

[Summary]
Multiple isolated phages were expected to bind to calmodulin. From 228 phages that read the sequence, 124 calmodulin-binding phages were recovered. It can be seen that the photopanning method obtained a selection efficiency of obtaining about 55% positive phage.

[Evaluation Experiment (17) Evaluation of Library Screening]
Whether the isolated calmodulin-binding protein was recovered in each photopanning cycle was confirmed by PCR specific to each gene. FIG. 24 shows a result of PCR using a phage solution amplified after panning as a template. Each sample information is shown in the upper part of the electrophoresis. “L” is a library template. In FIG. 25, the selected phage solution was appropriately diluted to make plaques appear, and SM buffer was added to prepare a phage solution. The results of amplification of each gene by PCR using this phage solution as a template are shown. The numerical value under each electrophoresis shows the approximate number of plaques that appeared on the used plate. In both figures, (9) and (7) are the results of the photopanning method, and (8) are the results of the conventional method.

[Summary]
In general, in the photopanning method ((7) and (9)), the detection frequency increases as the number of cycles increases for each gene. In the conventional method ((8)), most genes are not detected. Reproducibility is good when comparing two photopanning methods ((7) and (9)). The results of library screening are summarized in FIG.

  The present invention is useful in the field of drug discovery and antibody medicine related to proteins and peptides.

Schematic diagram of protein-displaying phage. Schematic explanatory drawing of the conventional panning method. Schematic explanatory drawing of the photopanning method of the present invention. Explanatory drawing of the photopanning method of this invention. The analysis result of HPLC in Example 3. The measurement result of the complex formation efficiency in Example 3. The analysis result by SDS-PAGE in the evaluation experiment (1) of Example 3. The analysis result by SDS-PAGE in the evaluation experiment (2) of Example 3. Explanatory drawing of the degree of concentration in one operation by the photopanning method. The result which confirmed the presence or absence of the calmodulin display phage in each dilution series in evaluation experiment (8). Evaluation Experiment (6) Sorting efficiency comparison result between the photopanning method and the conventional method. The schematic diagram of the experiment which isolates calmodulin display phage from the cDNA display phage library in Example 5 by the photopanning method and the conventional panning method. The amplification confirmation result by PCR method of the calmodulin gene in Example 5. Plaque hybridization results using the calmodulin gene as a probe in Example 5 (Note: The photographs do not include all plaques). Amplification confirmation result by PCR of calmodulin gene. Amplification confirmation result by PCR of calmodulin gene. Amplification confirmation result by PCR of calmodulin gene. Amplification confirmation result by PCR of calmodulin gene. Summary of Example 5. The result of confirming the presence or absence of calmodulin-binding peptide-displayed phage in each dilution series. Results of determination by plaque hybridization of the number of calmodulin-binding peptide-displayed phages in the phage solution captured by the photopanning method. Results of PCR amplification of the insert part. Results of analysis of No. 3 AC092953 in the calmodulin target database. Results of PCR using a phage solution amplified after panning as a template. Results of PCR amplification of each gene using a phage solution as a template. Summary of screening for calmodulin binding protein by photopanning method (invention). The screening method of the present invention can comprehensively analyze various clones with excellent reproducibility and selection efficiency.

Claims (23)

A photoreactive ligand having a photoreactive group and a protein-displayed phage or peptide-displayed phage are mixed in a liquid phase,
Irradiate an active light to the photoreactive group of the photoreactive ligand,
Immobilize the photoreactive ligand on the solid phase,
Washing the solid phase with immobilized photoreactive ligand,
A method for screening a protein or peptide that interacts with a photoreactive ligand, comprising recovering a protein-displayed phage or peptide-displayed phage bound to a photoreactive ligand immobilized on a solid phase. 2. The method according to claim 1, wherein the photoreactive ligand is a photoreactive peptide, a photoreactive protein, a photoreactive nucleic acid, or a photoreactive sugar chain. 3. The method according to claim 1, wherein the photoreactive group is a diazirine group. 4. The photoreactive ligand is immobilized on a solid phase using a biotin-avidin or streptavidin system, a His tag-nickel system, a boron-hydroxamic acid chelate system, or an antigen-antibody system. 2. The method according to item 1. (1) The photoreactive ligand has biotin and the solid phase is coated with avidin or streptavidin. (2) The photoreactive ligand has a His tag and the solid phase is coated with nickel. (3) the photoreactive ligand has a hydroxamic acid chelate, the solid phase is coated with boron, and (4) the photoreactive ligand has an antigen, and the solid phase is coated with an antibody. The method according to claim 4, wherein The method according to any one of claims 1 to 5, wherein the solid phase is a magnetic bead or a substrate. 7. The method according to claim 6, wherein the protein-displayed phage or peptide-displayed phage bound to the photoreactive ligand immobilized on the solid phase is recovered by recovering the magnetic beads. The method according to any one of claims 1 to 7, wherein the cleaning is performed using a surfactant. The method according to any one of claims 1 to 8, wherein the protein or peptide is identified by identifying the DNA of the recovered protein-displayed phage or peptide-displayed phage. Proliferating the recovered protein-displayed phage or peptide-displayed phage, and using the expanded protein-displayed phage or peptide-displayed phage,
Mixing with the photoreactive ligand according to claim 1 in a liquid phase, irradiation with active light, immobilization of the photoreactive ligand on a solid phase, washing of the solid phase, and immobilization on the solid phase Collecting at least one protein-displayed phage or peptide-displayed phage bound to the photoreactive ligand;
The method according to any one of claims 1 to 8. 11. The method according to claim 10, wherein the protein or peptide is identified by identifying the DNA of the recovered protein-displayed phage or peptide-displayed phage. A kit used to screen for proteins or peptides that interact with a photoreactive ligand,
The kit comprising a photoreactive ligand having a photoreactive group and a solid phase on which the photoreactive ligand is immobilized. The kit according to claim 12, wherein the photoreactive ligand is a photoreactive peptide, a photoreactive protein, a photoreactive nucleic acid, or a photoreactive sugar chain. The kit according to claim 12 or 13, wherein the photoreactive group is a diazirine group. The solid phase that immobilizes the photoreactive ligand is one that immobilizes the photoreactive ligand using biotin-avidin or streptavidin system, His tag-nickel system, boron-hydroxamic acid chelate system, or antigen-antibody system. The kit according to any one of claims 12 to 14. (1) The photoreactive ligand has biotin and the solid phase is coated with avidin or streptavidin. (2) The photoreactive ligand has a His tag and the solid phase is coated with nickel. (3) the photoreactive ligand has a hydroxamic acid chelate, the solid phase is coated with boron, and (4) the photoreactive ligand has an antigen, and the solid phase is coated with an antibody. The kit according to claim 15, which is any one of The kit according to any one of claims 12 to 16, wherein the solid phase is a magnetic bead or a substrate. The kit according to any one of claims 12 to 17, further comprising a cleaning agent for cleaning the solid phase on which the photoreactive ligand is immobilized. The kit according to any one of claims 12 to 18, which is used in the method according to any one of claims 1 to 11. A kit for producing a photoreactive ligand having a photoreactive group, comprising a compound having a photoreactive group, a ligand labeling substance, and a photoreaction mounting solution. The kit according to claim 20, wherein the compound having a photoreactive group is a phenyldiazirine compound represented by the following general formula (C).
(In the above formula, R ₆ is a halogen atom or a sulfonic acid ester residue, R ₇ is an alkylthiosulfonyl group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms, and R ₈ is H or an alkyl group having 1 to 6 carbon atoms, and n is an integer of 1 to 6) The kit according to claim 20 or 21, wherein the ligand labeling substance is biotin, a histidine tag (His tag), a hydroxamic acid chelate or an antigen. A kit for validation comprising a protein-displayed phage dilution solution and a photoreactive ligand solution.