Peptides

Abstract

To provide a target binding peptide.SOLUTION: A target-binding peptide obtained by a method comprising the steps of: bringing a peptide contained in a peptide library into contact with a target molecule; recovering a peptide bound to the target molecule; and preparing the recovered peptide by gene recombination.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a peptide, a derivative of the peptide, a peptide contained in the peptide or the derivative. Nucleotides corresponding to the nucleotides, vectors containing the nucleotides, said vectors and / or A method for producing a peptide and / or a method for producing a cell into which a nucleotide has been introduced, a ... and a method for producing the peptide and / or its derivative, and a peptide comprising the peptide and / or its derivative. Library, method for identifying peptides and / or their derivatives that bind to target molecules, and a method for producing a peptide and / or a derivative thereof that binds to a target molecule. or a derivative thereof binds to a target molecule, a nucleotide library comprising the peptide or a derivative thereof, the nucleotide, a composition comprising the vector or the cell, the peptide or a derivative thereof, the nucleic acid The present invention also relates to a reagent comprising said nucleotide, said vector or said cell, etc. [Background technology]

[0002] SPINK2(Serine Protease Inhibitor Kazal- Type 2) is the Kazal type with three disulfide bonds. It is a 7 kDa protein consisting of the α- and β-domains. It is expressed in the testis and seminal vesicles in humans. and functions as a trypsin / acrosin inhibitor ( Non-patent document 1).

[0003] In 1991, Winter et al. As a result of these findings, phage display has become a method for generating fully human antibodies. This has had a major impact on the development of antibody drugs (Non-Patent Document 2). By following these steps, we can develop display technologies such as phage display and ribosome display. The development of non-antibody scaffolds using spray technology is becoming more widespread. Non-antibody scaffolds are being developed Also called binding (affinity) protein, the variable region of an antibody It is an artificial protein with a CDR-like binding region. It has the ability to bind to protein X, allowing for protein-protein interactions, and also has the potential to improve productivity and immunogenicity. It has features such as tissue infiltration. ry (Dyax) (Patent Document 1) and anticalin (Pieris), etc. Although there is a desire for scientific or industrial advances in this field, the number of types is still limited, and there are few disease-related It is expected that a novel non-antibody scaffold for the fusion molecule will be created. (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication US2008 / 0020394 A1 (or Japanese Patent Application Publication 2007-524348) [Non-patent literature]

[0005] [Non-Patent Document 1] Chen T, Lee TR, Liang WG, Chang WS, Lyu PC. (2009) Identification of trypsin-inhibitory site and structure determination of human SPINK2 serine proteinase inhibitor. Proteins. 77(1):209-19. [Non-patent document 2] James D. Marks, Hennie R. Hoogenboom, Timothy P. Bonnert, John McCafferty, Andrew D. Griffiths, Greg Winter (1991) By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 222(3):581-97. [Non-patent document 3] Skerra A. (2007) Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol. 18:295-304. Summary of the Invention [Problem to be solved by the invention]

[0006] As a result of extensive investigations into SPINK2 and its variants, the inventors have found that SPINK2 We created a library containing peptides that exhibit high binding activity to molecules other than the endogenous target. From the library, peptides that exhibit high binding activity to target molecules other than endogenous targets are prepared. The present invention has been accomplished by isolating the peptide. [Means for solving the problem]

[0007] The present invention provides, for example, (1) A peptide selected from (i) or (ii) below: (i) a salt consisting of the 43rd base thymine to the 93rd base thymine in SEQ ID NO: 14 of the Sequence Listing The base sequence is replaced with a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. a peptide comprising an amino acid sequence encoded by a base sequence comprising: (ii) the amino acid sequences of amino acid numbers 2 to 8 and 10 shown in SEQ ID NO: 1 of the sequence listing; 1 to 5 amino acids other than 1 to 14 are substituted, deleted, added, and / or The peptide according to (i), having an amino acid sequence inserted therein; (2) The first to fifth, seventh, ninth and ninth residues counting from the amino terminus of SEQ ID NO: 1 in the sequence listing The 10th Xaa is any amino acid except cysteine ​​and proline. (1), (3) The 6th and 8th Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing are and any amino acid except cysteine. (4) The 11th Xaa counting from the amino terminal of SEQ ID NO: 1 in the sequence listing is tyrosine, serine, an amino acid selected from the group consisting of phenylalanine, leucine, and threonine (1) A peptide according to any one of (1) to (3). (5) The 12th Xaa from the amino terminal of SEQ ID NO: 1 in the sequence listing is asparagine, asparagine, from the group consisting of paragic acid, leucine, lysine, glutamine, alanine and glutamic acid The peptide according to any one of (1) to (4), wherein the amino acid is selected from the group consisting of: (6) Conservative amino acid substitutions are made in the hydrophobic amino acid group, the neutral hydrophilic amino acid group, and the acidic Amino acid groups, basic amino acid groups, amino acid groups that affect the direction of the main chain and aromatic amino acid group. The peptide according to any one of (1) to (5), (7) The first Xaa counting from the amino terminus of SEQ ID NO: 1 in the sequence listing is arginine, methionine an amino acid selected from the group consisting of riboflavin, leucine, tryptophan, and serine; A peptide according to any one of (1) to (6). (8) The second Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is threonine, arginine, or an amino acid selected from the group consisting of phenylalanine, tryptophan, and phenylalanine; A peptide according to any one of (1) to (7). (9) The third Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is arginine, histidine an amino acid selected from the group consisting of thiamin, tryptophan, serine, and phenylalanine The peptide according to any one of (1) to (8), (10) The fourth Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is tryptophan, Any of (1) to (9) is an amino acid selected from the group consisting of guanine and leucine. The peptide according to any one of the preceding claims.

[0008] (11) The fifth Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is glycine, arginine , leucine, histidine, tryptophan, methionine and tyrosine. The peptide according to any one of (1) to (10), wherein the amino acid is selected from the group consisting of: (12) The sixth Xaa from the amino terminal of SEQ ID NO: 1 in the sequence listing is asparagine, histidine, or PEG. The group consisting of arginine, proline, lysine, tryptophan, arginine, and aspartic acid The peptide according to any one of (1) to (11), wherein the amino acid is selected from (13) The seventh Xaa counting from the amino terminal of SEQ ID NO: 1 in the sequence listing is arginine, phenyl selected from the group consisting of alanine, tryptophan, leucine, alanine and glycine The peptide according to any one of (1) to (12), (14) The 8th Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is threonine, proline , asparagine, and serine. 3) A peptide according to any one of (15) The 9th Xaa from the amino terminal of SEQ ID NO: 1 in the sequence listing is tryptophan, methyl an amino acid selected from the group consisting of thiamin, tyrosine, and phenylalanine; The peptide according to any one of (1) to (14). (16) The 10th Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is glutamine or valine. , lysine, methionine, alanine, leucine, and asparagine. The peptide according to any one of (1) to (15), (17) The 11th Xaa counting from the amino terminal of SEQ ID NO: 1 in the sequence listing is tyrosine, phenyl (1) to (16) are amino acids selected from the group consisting of alanine and leucine. The peptide according to any one of (18) (1) The 12th Xaa from the amino terminus of SEQ ID NO: 1 in the Sequence Listing is lysine. (17) A peptide according to any one of (16) to (17). (19) SEQ ID NOs: 2 to 9 in the sequence listing (peptide numbers 1, 2, 6, 7, 12 to 14 in Figure 12) Any one of (1) to (18) containing an amino acid sequence represented by any one of (1) to (17). A peptide according to any one of claims 1 to 5, (20) The peptide according to any one of (1) to (19) is chemically modified and / or biologically modified. a derivative of said peptide which has been subjected to chemical modification;

[0009] (twenty one) A nucleotide according to any one of (i) to (iii) below: (i) A peptide encoding the amino acid sequence of any one of (1) to (19). nucleotides comprising nucleotides consisting of a base sequence corresponding to the nucleotide sequence; (ii) A peptide having an amino acid sequence according to any one of (1) to (19). nucleotides comprising a base sequence encoding the (iii) An amino acid sequence of a peptide according to any one of (1) to (19) Nucleotides consisting of a coding base sequence, (twenty two) A vector comprising the nucleotide according to (21). (twenty three) A cell into which the nucleotide according to (21) or the vector according to (22) has been introduced. (twenty four) Any one of (1) to (19) comprising the following steps (i) and (ii): Method for producing peptides: (i) culturing the cell according to (23); and (ii) recovering the peptide from the culture obtained in step (i); (twenty five) The peptide according to any one of (1) to (19) and / or the peptide according to (20) a peptide library comprising derivatives of a peptide; (26) The peptide and / or peptide derivative is prepared by the steps (i) and (ii) described in (24). (25) The light emitting device according to (25), characterized in that it is prepared by a method comprising: Braly, (27) In the library, the peptide or peptides that are phenotypes The derivative and the nucleotide corresponding to the genotype of the phenotype are directly The license according to (25) or (26), which is directly or indirectly linked to the Braly, (28) The library according to (27), wherein the nucleotide is the nucleotide according to (21). , (29) Phage display libraries, ribosome display libraries, etc. or a nucleic acid display library, (25) to (28). The library described, (30) A method for binding to a target molecule, comprising the steps of (i) and (ii) below: A method for identifying a peptide according to any one of (9) and (20) above or a peptide derivative according to (21): (i) A peptide or peptides contained in the library according to any one of (25) to (29). contacting the target molecule with a peptide derivative thereof; and (ii) recovering the peptide or peptide derivative that binds to the target molecule;

[0010] (31) A method for binding to a target molecule, comprising the steps of: (1) binding to a target molecule; A method for producing the peptide according to any one of (9) or the peptide derivative according to (20): (i) A peptide or peptides contained in the library according to any one of (25) to (29). contacting the target molecule with a peptide derivative thereof; and (ii) recovering the peptide or peptide derivative that binds to the target molecule; and (iii) contained in the peptide or peptide derivative recovered in (ii) above The peptide that binds to the target molecule can be synthesized by chemical synthesis, genetic recombination, or in vitro translation. preparing the (32) Any one of (1) to (19) comprising the following steps (i) and (ii): (20) Determine whether the peptide described in (20) or a derivative of the peptide described in (20) binds to a target molecule. How to set: (i) A test peptide according to any one of (1) to (19) or (20). contacting a test peptide derivative with the target molecule; and (ii) if the test peptide or test peptide derivative binds to the target molecule, determining that the peptide or test peptide derivative is positive; (33) A method for binding to a target molecule, comprising the steps of: (1) binding to a target molecule; A method for producing a derivative of the peptide according to any one of (9) and (20). : (i) A test peptide according to any one of (1) to (19) or (20). contacting a test peptide derivative with the target molecule; (ii) if the test peptide or test peptide derivative binds to the target molecule, determining that the peptide or test peptide derivative is positive; and (iii) The test peptide or peptide derivative was determined to be positive in step (ii). In this case, the peptide that binds to the target molecule and is contained in the peptide or peptide derivative is preparing the vector by genetic recombination or in vitro translation; (34) A nucleotide library comprising the nucleotide according to (21). (35) The nucleotide is a phagemid, cosmid or plasmid or a fragment thereof; 34) The library described in (36) If the nucleotide is present in a prokaryotic or eukaryotic cell, or on viral DNA or RNA, The nucleotide library according to (34) or (35), which is present in a virus particle or Lee, (37) A peptide according to any one of (1) to (19) and a derivative of the peptide according to (20). (21) The vector according to (22) or (23) a composition comprising: (38) A peptide according to any one of (1) to (19) and a derivative of the peptide according to (20). (21) The vector according to (22) or (23) a reagent comprising: (39) A peptide according to any one of (1) to (19), which binds to a predetermined target molecule. or a derivative of the peptide according to (20), (40) The peptide according to (39), wherein the target molecule is not an endogenous target of SPINK2. peptides or peptide derivatives,

[0011] (41) The peptide according to (39) or (40), wherein the target molecule is derived from a human. or a derivative of a peptide, (42) (39) to (41), wherein the endogenous target is trypsin and / or acrosin. A peptide or a derivative of a peptide according to any one of the preceding claims, (43) The peptide according to any one of (39) to (42), wherein the endogenous target is trypsin. derivatives of amides or peptides, (44) (39) to (43) a composition or reagent comprising: (45) A method for producing trypsin and / or acrosin comprising the following steps (i) to (iii): and binds to a serine protease other than Any one of (1) to (19) which inhibits peptide bond hydrolysis activity (hereinafter the same). A method for identifying a peptide according to (1) or a peptide derivative according to (20): (i) A peptide or peptides contained in the library according to any one of (25) to (29). contacting the serine protease with a peptide derivative thereof; (ii) recovering the peptide or peptide derivative that binds to the serine protease ; and, (iii) The peptide or peptide derivative is capable of undergoing proteolysis by the serine protease. determining the peptide or peptide derivative as positive if the activity is inhibited; (46) A method for producing a protein containing trypsin and / or acrosin, comprising the following steps (i) to (iv): binds to and inhibits the proteolytic activity of other serine proteases, (1) to (19 A method for producing the peptide according to any one of (19) and (20) or the peptide derivative according to (21): (i) A peptide or peptides contained in the library according to any one of (25) to (29). contacting the serine protease with a peptide derivative thereof; (ii) recovering the peptide or peptide derivative that binds to the serine protease and (iii) The peptide or peptide derivative recovered in step (ii) is When the proteolytic activity of protease is inhibited, the peptide or peptide derivative is determining that the result is positive; and (iv) The peptide or peptide derivative determined to be positive in step (iii) The peptides that inhibit the proteolytic activity of the serine protease are chemically synthesized and genetically engineered. preparing the vector by genetic recombination or in vitro translation; (47) Any one of (1) to (19) comprising the following steps (i) and (ii): The peptide or derivative of the peptide described in (20) is treated with trypsin and / or acetyltransferase. The proteolytic activity of serine proteases other than ricin (peptide bond hydrolysis activity: (hereinafter the same) to determine whether or not the following is inhibited: (i) A test peptide according to any one of (1) to (19) or (20). contacting a test peptide derivative with the serine protease; and (ii) The peptide or peptide derivative has the proteolytic activity of the serine protease. determining the peptide or peptide derivative as positive if the activity is inhibited; (48) A method for producing trypsin and / or acrosin comprising the following steps (i) to (iii): Any of (1) to (19) which inhibits the proteolytic activity of serine proteases other than A method for producing a derivative of the peptide according to any one of (19) and (20): (i) A test peptide according to any one of (1) to (19) or (20). contacting a test peptide derivative with the serine protease; (ii) The peptide or peptide derivative has the proteolytic activity of the serine protease. determining the peptide or peptide derivative as positive if the peptide or peptide derivative inhibits the activity; and (iii) The peptide or peptide derivative determined to be positive in step (ii) The peptide that inhibits the proteolytic activity of the serine protease is genetically modified. preparing the vector by in vitro translation or in vitro translation; and (49) A peptide selected from (i) or (ii) below: (i) Contains the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and is one of the following (a) to (d): Certain peptides; (a) The first to fifth, seventh, ninth and ninth residues counting from the amino terminus of SEQ ID NO: 1 in the sequence listing The 10th Xaa is any amino acid except cysteine ​​and proline. , (stomach) The 6th and 8th Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing are , any amino acid except cysteine, (cormorant) The 11th Xaa counting from the amino terminal of SEQ ID NO: 1 in the sequence listing is tyrosine, serine, an amino acid selected from the group consisting of phenylalanine, leucine, and threonine , (workman) The 12th Xaa from the amino terminal of SEQ ID NO: 1 in the sequence listing is asparagine, asparagine, from the group consisting of paragic acid, leucine, lysine, glutamine, alanine and glutamic acid The amino acids of choice are: and, (ii) In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, the 1st amino acid from the amino terminal one to five amino acids other than Xaa at positions 1 to 12 are conservative amino acid substitutions; Peptides containing amino acid sequences comprising deletions, additions and / or insertions; (50) Conservative amino acid substitutions are made in the hydrophobic amino acid group, the neutral hydrophilic amino acid group, and the acidic Amino acid groups, basic amino acid groups, amino acid groups that affect the direction of the main chain and aromatic amino acid group. The peptide according to (49),

[0012] (51) The peptide according to (49) or (50), which is any one of the following (A) to (L): (a) The first Xaa counting from the amino terminus of SEQ ID NO: 1 in the sequence listing is arginine, methionine an amino acid selected from the group consisting of riboflavin, leucine, tryptophan, and serine; (stomach) The second Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is threonine, arginine, or an amino acid selected from the group consisting of phenylalanine, tryptophan, and phenylalanine; (cormorant) The third Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is arginine, histidine an amino acid selected from the group consisting of thiamin, tryptophan, serine, and phenylalanine That is, (workman) The fourth Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is tryptophan, an amino acid selected from the group consisting of guanine and leucine; (E) The fifth Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is glycine, arginine , leucine, histidine, tryptophan, methionine and tyrosine. The amino acid of choice is (mosquito) The sixth Xaa from the amino terminal of SEQ ID NO: 1 in the sequence listing is asparagine, histidine, or PEG. The group consisting of arginine, proline, lysine, tryptophan, arginine, and aspartic acid is an amino acid selected from (tree) The seventh Xaa counting from the amino terminal of SEQ ID NO: 1 in the sequence listing is arginine, phenyl selected from the group consisting of alanine, tryptophan, leucine, alanine and glycine It is an amino acid that (nine) The 8th Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is threonine, proline , asparagine, and serine; (K) The 9th Xaa from the amino terminal of SEQ ID NO: 1 in the sequence listing is tryptophan, methyl an amino acid selected from the group consisting of onine, tyrosine and phenylalanine; (Ko) The 10th Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is glutamine or valine. , lysine, methionine, alanine, leucine, and asparagine. It is an amino acid that (sa) The 11th Xaa counting from the amino terminal of SEQ ID NO: 1 in the sequence listing is tyrosine, phenyl an amino acid selected from the group consisting of alanine and leucine, (C) The 12th Xaa from the amino terminus of SEQ ID NO: 1 in the sequence listing is lysine. (52) The peptide according to any one of (49) to (51) is chemically modified and / or synthesized. a derivative of said peptide which has been subjected to a chemical modification; (53) The peptide according to any one of (49) to (51), which binds to a predetermined target molecule. a peptide or a derivative of the peptide according to (52), and, (54) The peptide according to (53), wherein the target molecule is not an endogenous target of SPINK2. peptides or peptide derivatives, The present invention relates to, but is not limited to, the following: [Effects of the Invention]

[0013] The present invention provides a peptide label useful for selecting peptides that bind to a desired target molecule. It is possible to provide a library. [Brief explanation of the drawings]

[0014] [Figure 1] SPINK2 mutants (polyclonal clones) obtained by panning against the target molecule α-chymotrypsin were displayed on phages, and their binding to the target molecule was confirmed by ELISA. BSA was used as a negative control molecule. [Figure 2] SPINK2 mutants (polyclonal clones) obtained by panning against the target molecule, plasma kallikrein, were displayed on phages and their binding to the target molecule was confirmed by ELISA. BSA was used as a negative control molecule. [Figure 3]SPINK2 mutants (polyclones) obtained by panning against the target molecule hEGFR / Fc were displayed on phages, and their binding to the target molecule was confirmed by ELISA. BSA was used as a negative control molecule. [Figure 4] SPINK2 mutants (polyclonal) obtained by panning against the target molecule hHER2 / Fc were displayed on phages, and their binding to the target molecule was confirmed by ELISA. BSA was used as a negative control molecule. [Figure 5] The molecular state of the α-chymotrypsin binding peptide (single clone) expressed and purified in E. coli was analyzed by SDS-PAGE under reducing conditions. [Figure 6] The molecular state of the α-chymotrypsin binding peptide (single clone) expressed and purified in E. coli was analyzed by SDS-PAGE under non-reducing conditions. [Figure 7] The binding affinity of eight α-chymotrypsin-binding peptides expressed and purified in E. coli was quantitatively measured by ELISA. [Figure 8] The binding affinity of α-chymotrypsin-binding peptides No. 2 and 6 (clone 2 and 6) expressed and purified in E. coli was quantitatively measured by ELISA. [Figure 9] The target specificity of α-chymotrypsin-binding peptide No. 2 (clone 2) expressed and purified in E. coli was confirmed by ELISA. [Figure 10] The target specificity of α-chymotrypsin-binding peptide No. 6 (clone 6) expressed and purified in E. coli was confirmed by ELISA. [Figure 11] Quantitative measurement of the chymotrypsin inhibitory activity of α-chymotrypsin-binding peptides Nos. 2 and 6 (clone 2 and 6) expressed and purified in E. coli. [Figure 12] Amino acid sequences (SEQ ID NOs: 2 to 9) of the random regions of eight SPINK2 mutants (α-chymotrypsin-binding peptide numbers 1, 2, 6, 7, 12 to 14, and 17). [Figure 13] Amino acid sequence of the random region of the diverse peptide (SEQ ID NO: 1). Amino acid numbers 2 to 8 and 10 to 14 represent arbitrary amino acids. [Figure 14] Nucleotide sequence of fragment 1 (SEQ ID NO: 10) [Figure 15-1] Nucleotide sequence of pCANTAB 5E (continued in Figure 15-2) [Figure 15-2] The base sequence of pCANTAB 5E (the underlined part is the same as SEQ ID NO: 11, and is the base sequence of "Fragment 2": continued in Figure 15-3) [Figure 15-3] Nucleotide sequence of pCANTAB 5E (continued from Figure 15-4) [Figure 15-4] Nucleotide sequence of pCANTAB 5E [Figure 16] Nucleotide sequence of fragment 3 (SEQ ID NO: 12) [Figure 17] Nucleotide sequence of fragment 5 (SEQ ID NO: 13) [Figure 18] Nucleotide sequence encoding the amino acid sequence of SPINK2 (SEQ ID NO: 14) [Figure 19] Amino acid sequence encoded by the base sequence shown in Figure 18 (SEQ ID NO: 15) [Figure 20-1] Base sequence of PCR template DNA including fragments 1 to 5 (SEQ ID NO: 16: continued in Figure 20-2) [Figure 20-2] Base sequence of PCR template DNA including fragments 1 to 5 (SEQ ID NO: 16: continued) DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to peptides, peptide derivatives, peptide libraries, nucleotides, vectors, and the like. Methods for producing tethers, cells, peptides and / or derivatives thereof, peptides having desired properties Method for identifying peptides and / or derivatives thereof, peptides and / or derivatives thereof having desired properties, and determining whether a test peptide or its derivative binds to a target molecule. The present invention provides methods, nucleotide libraries, compositions, reagents, and the like. However, the present invention is not limited to these embodiments.

[0016] 1. Peptides The present invention provides peptides.

[0017] The term "peptide" as used herein also encompasses "polypeptide" and "protein." In addition, in the present invention, such a "peptide" is a "peptide contained in a peptide derivative." is also included in the meaning.

[0018] In one embodiment of the present invention, the peptide has the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. The peptide contains the amino acid sequence shown in SEQ ID NO: 1. The 1st to 12th Xaa counting from the amino terminus are each any amino acid, Preferably, it is any amino acid except cysteine.

[0019] In a more preferred embodiment of the present invention, the amino acid sequence of the peptide of the present invention is , the first to fifth amino acids counting from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, the 7th, 9th and 10th Xaa (the 2nd to 6th, 8th and 11th Xaa of SEQ ID NO: 1); and 12th amino acids, respectively) are any amino acid except cysteine ​​and proline. The 6th and 8th Xaa from the amino terminus (SEQ ID NO: 1) are any amino acids; The amino acids corresponding to the 7th and 10th amino acids of The 11th Xaa from the amino terminus (the 13th amino acid in SEQ ID NO: 1) The amino acids (corresponding to the amino acids) are tyrosine, serine, phenylalanine, leucine, and threonine the 12th amino acid from the amino terminus is selected from the group consisting of Xaa( (corresponding to the 14th amino acid of SEQ ID NO: 1) is asparagine, aspartic acid, leucine, an amino acid selected from the group consisting of lysine, lysine, glutamine, alanine and glutamic acid; and Xaa at positions 1 to 12 counting from the amino terminus is an amino acid as described in this paragraph. The amino acids selected from each of the groups listed above may be replaced with conservative amino acid substitutions (as in other embodiments of the present invention). (as detailed in part 1).

[0020] In a more preferred embodiment of the present invention, the amino acid sequence of the peptide of the present invention is In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, the first X from the amino terminal is aa (corresponding to the second amino acid in SEQ ID NO: 1) is arginine, methionine, leucine amino acids selected from the group consisting of thiamin, tryptophan, and serine; The second Xaa counting from the end (corresponding to the third amino acid in SEQ ID NO: 1) is threonine an amino acid selected from the group consisting of arginine, tryptophan, and phenylalanine; the third Xaa from the amino terminus (the fourth amino acid in SEQ ID NO: 1); The corresponding amino acids are arginine, histidine, tryptophan, serine, and phenylalanine. The fourth Xaa( The amino acid sequence corresponding to the fifth amino acid in SEQ ID NO: 1 is composed of tryptophan, arginine, and leucine. Xaa is an amino acid selected from the group consisting of: (corresponding to the sixth amino acid of SEQ ID NO: 1) is glycine, arginine, leucine, hyaluronic acid, an amino acid selected from the group consisting of thiazolidine, tryptophan, methionine and tyrosine; The sixth Xaa from the amino terminus (corresponding to the seventh amino acid in SEQ ID NO: 1) The amino acids that make up the amino acids are asparagine, histidine, proline, lysine, tryptophan, and arginine. and aspartic acid; In addition, the seventh Xaa (corresponding to the eighth amino acid of SEQ ID NO: 1) is arginine, phenyl Selected from the group consisting of hydroxyl alanine, tryptophan, leucine, alanine and glycine the 8th Xaa from the amino terminus (the 10th Xaa in SEQ ID NO: 1); (corresponding to the amino acids threonine, proline, asparagine and serine) the 9th Xaa from the amino terminus (SEQ ID NO: 1) is an amino acid selected from the group The amino acids that make up the 11th amino acid in phenylalanine; the 10th amino acid from the amino terminus is selected from the group consisting of Xaa (corresponding to the 12th amino acid of SEQ ID NO: 1) is glutamine, valine, Selected from the group consisting of lysine, methionine, alanine, leucine and asparagine The 11th Xaa from the amino terminus (the 13th Xaa in SEQ ID NO: 1) amino acid) is selected from the group consisting of tyrosine, phenylalanine and leucine The 12th Xaa from the amino terminus (number 14 in SEQ ID NO: 1) is the amino acid The first amino acid from the amino terminus is lysine; The second Xaa is an amino acid selected from each group described in this paragraph, and is a conservative amino acid. It may be substituted (as detailed elsewhere in this specification).

[0021] Furthermore, in a preferred embodiment of the present invention, the peptide of the present invention is SEQ ID NO: 1 In addition to the amino acid sequence represented by the formula (I), there is provided an amino acid sequence, which is directly or in one or more is a nucleotide sequence consisting of two or more amino acids, the first base of SEQ ID NO: 14 in the sequence listing, guanine, or Amino acid sequence encoded by the base sequence consisting of the 4th base cytosine to the 42nd base thymine The amino acid sequence is located directly or flanked by one or more amino acids on the carboxyl terminal side. The nucleotide sequence is composed of the 94th base guanine and the 189th base cytosine of SEQ ID NO: 14 in the sequence listing. Each of the peptides may contain an amino acid sequence encoded by the base sequence. The amino acid sequence of the nucleotide sequence is from the first base guanine to the second base of SEQ ID NO: 14 in the sequence listing. The aspartic acid encoded by the third base, thymine, is not included in the corresponding position. In addition, the amino acid sequence of such a peptide may further include any other amino acid. Such peptides may include, for example, the peptides described in Example 1 below. The peptides contained in the randomly mutated SPINK2 library prepared in the above The peptides selected from the library in Example 3 and the amino acid sequences of the random regions were determined. Examples of suitable anti-inflammatory drugs include, but are not limited to, thiamin ...

[0022] In one embodiment of the present invention, the amino acid sequence of the peptide is The amino acid sequence shown in SEQ ID NO: 1 is Amino acids other than a may be substituted, deleted, added and / or inserted. In the amino acid sequence, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or an amino acid corresponding to said sequence Substitutions, deletions, or additions other than Xaa at positions 1 to 12 from the amino terminus of the amino acid sequence The number of added and / or inserted amino acids may be 1 to 10, and the lower limit is The upper limit is 10, 9, 8, 7, 6, 5, 4, 3, 2. There should be at least 1 amino acid substitution, and such amino acid substitutions should preferably be conservative amino acid substitutions.

[0023] In such an amino acid sequence, the amino acid sequence shown in SEQ ID NO: 1 or a sequence corresponding to said sequence The first to twelfth Xaa's counting from the amino terminus of the amino acid sequence are each any Any amino acid is preferred, but any amino acid other than cysteine ​​is preferred, and more preferably or an amino acid selected from the above groups, or the amino acid is a conservative amino acid substitution. It has been replaced.

[0024] "Conservative amino acid substitutions" "Titation" means substitution with a functionally equivalent or similar amino acid. Conservative amino acid substitutions in a peptide result in a silent change in the amino acid sequence of the peptide. For example, one or more amino acids of a similar polarity act functionally equivalently, This results in a static change in the amino acid sequence of the peptide. Generally, substitutions within a group are It can be considered to be conserved in structure and function. As is obvious, the role played by a particular amino acid residue is determined by the three-dimensional structure of the molecule containing that amino acid. For example, cysteine ​​residues can be determined in the reduced form (cysteine). It takes on an oxidized (disulfide) form that is less polar than the (all) form. The long aliphatic portion of the arginine side chain provides important structural and functional features. It can also be composed of side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine). can contribute to ion-aromatic interactions or cation-pi interactions. In this context, amino acids with these side chains are classified as belonging to the acidic or nonpolar groups. Substitutions can be structurally and functionally conservative. Residues such as disulfide forms may have a direct effect on the main chain conformation. and often cannot be replaced without structural distortion.

[0025] Conservative amino acid substitutions are specific substitutions based on side chain similarity (Leni), as shown below. L. Lehning, Biochemistry, 2nd revised edition, 1975, pp. 73-75: er, Biochemistry, 2 nd edition, pp73-75, Worth Publisher, New York (1975) and typical placement Includes exchange.

[0026] (1) Nonpolar amino acid group: alanine (hereinafter referred to as "Ala" or simply "A") valine (hereinafter referred to as "Val" or simply "V"), leucine (hereinafter referred to as "Leu" or simply "V"), or simply "L"), isoleucine (hereinafter referred to as "Ile" or simply "I"), Proline (hereinafter referred to as "Pro" or simply "P"), phenylalanine ("Phe") or simply "F"), tryptophan (hereinafter referred to as "Trp" or simply "W" ), methionine (hereinafter referred to as "Met" or simply "M") (2) Uncharged polar amino acid group: glycine (hereinafter referred to as "Gly" or simply "G") Serine (hereinafter referred to as "Ser" or simply "S"), threonine (hereinafter referred to as "Th") cysteine ​​(hereinafter referred to as "Cys" or simply "C"), ), tyrosine (hereinafter referred to as "Tyr" or simply "Y"), asparagine (hereinafter referred to as "A sn" or simply "N"), glutamine (hereinafter referred to as "Gln" or simply "Q") vinegar) (3) Acidic amino acid group: aspartic acid (hereinafter referred to as "Asp" or simply "D") Glutamic acid (hereinafter referred to as "Glu" or simply "E") (4) Basic amino acid group: lysine (hereinafter referred to as "Lys" or simply "K"), Arginine (hereinafter referred to as "Arg" or simply "R"), histidine (hereinafter referred to as "His " or simply "H")

[0027] Furthermore, naturally occurring amino acids are divided into the following groups based on the properties of their common side chains: It can be divided into loops. (1) Hydrophobic amino acid group: Norleucine, Met, Al a, Val, Leu, Ile (2) Neutral hydrophilic amino acid group: Cys, Ser, Thr, Asn, Gln (3) Acidic amino acid group: Asp, Glu (4) Basic amino acid group: His, Lys, Arg (5) Group of amino acids that influence the direction of the main chain: Gly, Pro (6) Aromatic amino acid group: Trp, Tyr, Phe Examples of conservative substitutions are shown below, but the conservative amino acid substitutions of the present invention are not limited to these. It's not something like that.

[0028] Ala, for example, Val, Leu, Ile, Met, Norleucine, Pro, Phe , can be substituted for Trp. Arg can be substituted for, for example, Lys, His. Asn can be substituted with, for example, Cys, Ser, Thr, Gln, Tyr, or Gly. Asp may, for example, be substituted for Glu. Cys can be substituted for, for example, Gly, Ser, Thr, Tyr, Asn, or Gln. Gln can be substituted for, for example, Gly, Ser, Thr, Cys, Tyr, or Asn. Glu, for example, can be substituted for Asp. Gly can be, for example, Ser, Cys, Thr, Tyr, Asn, Gln, Pro, As p, can be substituted for Glu. His can be substituted for, for example, Lys, Arg. Ile is, for example, Leu, Val, Met, Pro, Ala, Phe, Trp, Nor It may be substituted for leucine. Leu can be, for example, norleucine, Ile, Val, Pro, Met, Ala, Phe , Trp, Met can be substituted Lys can be substituted for, for example, Arg, His. Met can be, for example, Ala, Val, Leu, Phe, Ile, Pro, Trp, Nor It may be substituted for leucine. Norleucine is, for example, Met, Ala, Val, Leu, Ile, Pro, Phe , can be substituted for Trp. Phe can be, for example, Trp, Leu, Val, Ile, Ala, Tyr, Pro, Me It can be replaced with t. Pro is, for example, Ala, Val, Leu, Ile, Phe, Trp, Met, Glu It can be substituted for y. Ser can be substituted for, for example, Thr, Cys, Asn, Gln, Gly, or Tyr. Thr can be substituted with, for example, Val, Ser, Gly, Cys, Tyr, Asn, or Gln. possible. Trp is, for example, Tyr, Phe, Ala, Val, Leu, Ile, Pro, Me It can be replaced with t. Tyr can be, for example, Gly, Cys, Asn, Gln, Trp, Phe, Thr, or Se. Can be substituted for r. Val can be, for example, Ile, Leu, Met, Trp, Phe, Ala, or norleucine. , can be substituted for Pro.

[0029] The amino acid sequence of the peptide of the present invention is composed of such amino acids. The amino acid sequence contained in the amino acid sequence (corresponding to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing) Examples of the random region include the following, but the peptides of the present invention The amino acid sequence of the domain is not limited to these.

[0030] CRTRW GNRCT WQYKP VC (SEQ ID NO: 2 in the Sequence Listing: α-K in Figure 12 trypsin-binding peptide no. 1) CMRHR RHFCT MVYKP VC (SEQ ID NO: 3 in the Sequence Listing: α-K in Figure 12 Trypsin-binding peptide no. 2 CRRWL LPWCT YKYKP VC (SEQ ID NO: 4 in the Sequence Listing: α-K in Figure 12 Trypsin-binding peptide no. 6 CLWRR HKLCP FKFKP VC (SEQ ID NO: 5 in the Sequence Listing: α-K in Figure 12 Trypsin-binding peptide no. 7 CWRSW RWACP YMYKP VC (SEQ ID NO: 6 in the Sequence Listing: α-K in Figure 12 Trypsin-binding peptide no. 12 CWFFR WRWCN WALKP VC (SEQ ID NO: 7 in the Sequence Listing: α-K in Figure 12 Trypsin-binding peptide 13 CSTWR MWGCP WLYKP VC (SEQ ID NO: 8 in the Sequence Listing: α-K in Figure 12 Trypsin-binding peptide no. 14 CWRRW YDRCS FNLKP VC (SEQ ID NO: 9 in the Sequence Listing: α-K in Figure 12 Trypsin-binding peptide no. 17

[0031] In the present invention, amino acids may be L-amino acids, D-amino acids, or mixtures thereof (DL -amino acids), but refers to L-amino acids unless otherwise specified.

[0032] In the present invention, the amino acids include amino acids other than those mentioned above (hereinafter, for convenience, referred to as "abnormal amino acids"). The unusual amino acids may be, for example, those found in natural peptides or Selenocysteine, N-formylmethionine, pyrrolidine, pyridinium, and cysteine ​​found in proteins O-glutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, Phosphoserine, desmosine, β-alanine, sarcosine, ornithine, creatine, γ-alanine Aminobutyric acid, opine, theanine, tricholominic acid, kainic acid, domoic acid, acromelic acid Examples of unnatural amino acids include Ac-amino acids and Boc-amino acids. N-terminal protected amino acids such as Fmoc-amino acids, Trt-amino acids, and Z-amino acids, Amino acid t-butyl ester, benzyl ester, cyclohexyl ester, fluorenyl C-terminal protected amino acids such as esters, diamines, ω amino acids, β amino acids, γ amino acids, Examples include Tic derivatives of amino acids, and other amino acids including aminophosphonic acid. It can be, but is not limited to, these.

[0033] The peptides of the present invention can be prepared by chemical synthesis, genetic recombination, in vitro translation, or other methods to prepare peptides or proteins. The present invention can be prepared by a method well known to those skilled in the art. Peptides identified or selected from libraries etc. can also be prepared by these methods. This can be done.

[0034] As a chemical synthesis method, for example, t-butoxycarbonyl nyl:Boc method, 9-Fluorenylmethoxycarbonyl (9-Fluorenylm ethoxycarbonyl (Fmoc) method, but not limited to these. The Fmoc method has mild deprotection conditions and is easy to cleave the peptide from the resin. It has the advantage of being easy to extract (Fmoc solid phase p eptide synthesis: a practical approach, ed. by WC Chan, PD White Eds., Oxf ord University Press, New York, 2000.).

[0035] In the present invention, the term "derivative of a peptide" and "peptide derivative" refers to a peptide of the present invention. It means that the peptide has been chemically or biologically modified. In or on the peptide of the present invention, chemical reactions, i.e., atom-atom bonds, may occur. This means that the original peptide is changed into a substance different from the original peptide by the formation of bonds or cleavage. Biological modifications are modifications that induce biological reactions, including cleavage, in or on the peptides of the present invention. That is, proteins (enzymes, cytokines, etc.) derived from living organisms, nucleic acids (ribozymes, etc.), cells, using tissues, organs or non-human individuals, or relying on their direct or indirect effects This means that a substance different from the original peptide is produced.

[0036] Such a "derivative" is not particularly limited as long as it is a substance different from the original peptide. However, for example, those containing naturally occurring or artificially created sugar chains, polyethylene including polymers such as PEG (polyethylene glycol), synthetic or natural compounds Those labeled with the above, those containing the portion necessary for immobilization, and those containing a signal peptide at the amino terminal. those containing tags for use in purification or isolation; Amino acids and amino acid sequences derived from vectors suitable for panning, expression, etc. The base sequence encoding the peptide to avoid a defect or to introduce a restriction enzyme site The amino acids, amino acid sequences, and phenotypes of peptides that are produced by modifying and the genotype corresponding to said phenotype is directly or indirectly linked, and , and a combination of two or more of them.

[0037] The peptide derivatives of the present invention can be produced by chemical reactions, biochemical reactions, Methods for chemically or biologically modifying peptides and proteins, such as post-translational modifications, are well known to those skilled in the art. The library of the present invention can be prepared by a well-known method. Derivatives of peptides identified or selected from the above can also be prepared by these methods. In addition, such translation can also be achieved by genetic recombination using cells that have the desired post-translational modification ability. It is possible to prepare peptide derivatives of the present invention that have undergone post-translational modifications. By adding modified amino acids to the translation system, the peptides of the present invention containing the modified amino acids can be produced. Derivatives can be prepared.

[0038] As a method of PEGylation, for example, a peptide or protein and N-hydroxysuc Examples include a method of reacting with cimide ester (NHS)-PEG. However, it is not limited to this.

[0039] In a preferred embodiment, the peptides of the present invention and their derivatives bind to target molecules (other peptides of the present invention). The present invention binds to a predetermined target molecule (described in detail in the section below). The peptide of the present invention can be used as a reagent for testing various diseases for which the target molecule can be a marker. As described above, the present invention provides a peptide having a sequence, which is included in the amino acid sequence of the peptide. The first to twelfth amino acids from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 in the table Substitution or deletion of one or several (several being an arbitrary integer number of 1 to 10) amino acids other than Xaa, The peptides include those having amino acid sequences with additions and / or insertions. In its preferred embodiment, the peptide binds to a target molecule, and such target molecule is predetermined. It is preferable that

[0040] The peptides of the present invention and their derivatives can be in the form of, for example, an isolated form. (lyophilized preparation, solution, etc.), forms bound to other molecules (solid phase forms, fusion proteins, Physical assemblies including other peptides ( On the surface of cells (such as E. coli or yeast cells) a form in which the gene is expressed or displayed (synonymous with display) in a cell of the present invention, or a virus Examples include forms expressed or presented (synonymous with display) on particles, It is not limited to these, and any form suitable for the purpose of use, storage, etc. may be selected. It is possible.

[0041] 2. Nucleotides The present invention provides nucleotides.

[0042] In the present invention, a "nucleotide" refers to a mononucleotide, an oligonucleotide, or A polynucleotide is also called a "nucleic acid," a "nucleic acid molecule," or a "gene." Examples of the nucleotides include DNA, cDNA, RNA, mRNA, cRNA, Examples include probes, oligonucleotides, polynucleotides, primers, vectors, etc. The nucleotide of the present invention can be, but is not limited to, a single It can be any of a single strand nucleotide, a double strand nucleotide, and an aggregate of three or more nucleotides. may be a hybrid single-stranded nucleotide consisting of DNA and RNA, double-stranded consisting of a nucleotide and its complementary strand; hybrids consisting of single-stranded DNA and single-stranded RNA; double-stranded RNA, single-stranded nucleotides that may have double-stranded structures within the molecule, etc. Furthermore, the nucleotides of the present invention may be any of naturally occurring bases or mononucleotides. Contains one or more (artificially created) bases or mononucleotides other than nucleotides It's okay to do that.

[0043] A preferred nucleotide of the present invention is a nucleotide encoding the amino acid sequence of the peptide of the present invention. a nucleotide comprising a nucleotide sequence encoding the peptide of the present invention; Examples of the nucleotide sequence include a nucleotide sequence encoding an amino acid sequence corresponding to the nucleotide sequence. Such a suitable nucleotide sequence encodes the amino acid sequence of the peptide of the present invention. It may contain base sequences other than the base sequence and / or non-nucleotide moieties, and may be chemically or may be biologically modified (as described elsewhere in this invention). All of these are included in the term "nucleotide."

[0044] The nucleotide of the present invention encodes the amino acid sequence of the peptide of the present invention. It also includes nucleotides consisting of a base sequence.

[0045] In the present invention, the amino acid sequence of the peptide of the present invention is a nucleotide salt. When encoded by part or all of a base sequence, such nucleotides are referred to as "(such) nucleotides." nucleotide corresponding to a (similar) peptide" and such a peptide is called a "(similar) nucleotide" These peptides are called "peptides corresponding to the nucleotides."

[0046] Examples of nucleotides corresponding to the peptides of the present invention include those having the amino acid sequence of the peptides of the present invention. a nucleotide comprising a nucleotide sequence encoding an amino acid sequence of Nucleotides comprising a base sequence encoding the amino acid sequence of the peptide of the present invention nucleotides consisting of a base sequence encoding the amino acid sequence of the peptide of the present invention, etc. These can include, but are not limited to:

[0047] The peptide corresponding to the nucleotide of the present invention is a peptide having the base sequence of the nucleotide of the present invention. A peptide comprising a peptide consisting of an amino acid sequence encoded by a part or all of The amino acid sequence encoded by a part or all of the base sequence of the nucleotide of the present invention a peptide comprising a sequence encoded by a part or the whole of the base sequence of the nucleotide of the present invention; peptides consisting of the amino acid sequence to be synthesized, or derivatives of any one of these peptides, These can include, but are not limited to:

[0048] In the present invention, the expression "genotype corresponding to phenotype" also refers to "nucleotide corresponding to peptide." Similarly, the expression "phenotype corresponding to genotype" is also used as " The term "peptide corresponding to a nucleotide" is used interchangeably with "nucleotide corresponding to a peptide."

[0049] Chemical or biological modifications of the nucleotides of the present invention include the peptides of the present invention. In such cases, such modifications are included within the meaning of the "derivatives of peptides" of the present invention described above.

[0050] Among the above-mentioned preferred nucleotides, the more preferred nucleotides of the present invention are those that are A nucleic acid sequence encoding the amino acid sequence of the peptide of the present invention that binds to the molecule. The peptide of the present invention binds to a target molecule. a nucleotide comprising a base sequence encoding a target molecule; Examples include nucleotides consisting of a base sequence that codes for the amino acid sequence of a peptide. This can be done.

[0051] In the present invention, when designing a base sequence encoding an amino acid sequence, One or more codons corresponding to the corresponding amino acids can be used. The base sequence encoding a single amino acid sequence of a gene or protein may have multiple variations. In selecting such codons, the genotype corresponding to the peptide, i.e., The codon usage of the cell (host cell) into which the nucleotide containing the base sequence is introduced n usage), or by selecting appropriate codons depending on the frequency or usage of multiple codons. The ratio can be adjusted appropriately. For example, when E. coli is used as a cell (host cell), If this is the case, the base sequence can be designed using codons that are frequently used in E. coli. Cut.

[0052] The nucleotide of the present invention can be produced by a method for producing nucleotides, such as chemical synthesis or genetic recombination. These can be prepared by methods well known to those skilled in the art. The nucleic acids corresponding to the peptides recovered (including selection, enrichment, and isolation) by the identification method of the present invention are Nucleotides can also be prepared by these methods.

[0053] The nucleotide of the present invention can take, for example, an isolated form (lyophilized form). (preparation, solution, etc.), in a form bound to other molecules (immobilized, etc.), containing the nucleotide Recombinant vector (the vector of the present invention), the nucleotide or the vector introduced into a cell a cell (the cell of the present invention), a form contained in a virus or virus particle (the vector of the present invention) a physical assembly containing other nucleotides, etc. (including forms included as the nucleotides of the present invention) Examples of suitable libraries include, but are not limited to, those listed below. The form can be freely selected to suit the purpose of use, storage, etc.

[0054] 3. Vector The present invention provides a recombinant vector (hereinafter also simply referred to as "vector").

[0055] The vector of the present invention comprises a nucleotide of the present invention and is capable of expressing the nucleotide of the present invention in a cell. There is no particular limitation as long as it is a means for introducing the gene into a microorganism or an individual, but it is preferably a method using a Examples of such nucleic acid vectors include nucleotides, cosmids, and plasmids.

[0056] The vectors of the present invention may be viruses or viral vectors that infect prokaryotic or eukaryotic cells. It may also be a ctor.

[0057] In the present invention, a "phagemid" is a single-stranded bacterial vector that contains a plasmid replication origin as well as a It refers to a bacterial plasmid containing a second origin of replication derived from a phage. Cells carrying the lysamide are transformed with M13 or a similar helper bacteriophage. Upon superinfection, the phagemid can replicate via a single-stranded replication mode. That is, the single-stranded fragment is contained in the infectious particle coated with the bacteriophage coat protein. The phagemid DNA is then packaged. Clone the phagemid as a double-stranded DNA plasmid into bacteria and culture the cells superinfected They can be formed from the supernatant as bacteriophage-like particles. The phage-like particles are then transferred to bacteria with F-pili to infect the cells with the corresponding DNA. By injecting it into bacteria, the particles can reform themselves as plasmids.

[0058] Nucleotides corresponding to the peptides of the present invention and bacteriophage coat proteins (co A fusion gene comprising a nucleotide sequence encoding a nucleotide sequence of a target gene is inserted into the phagemid. By infecting bacteria with the peptide and culturing the cells, the peptide is transferred to the bacteria or phage-like The protein is expressed or presented (synonymous with display) on the particle, or interacts with the coating protein. It can be produced as a fusion protein in a phage particle or in the culture supernatant of the bacterium.

[0059] For example, nucleotides and bacteriophage coated proteins corresponding to the peptides of the invention The fusion gene containing the protein gene gpIII was inserted into a phagemid and transformed into M13 or a similar helper phage, the peptide and and the coating protein, and the resulting fusion protein is produced in the culture supernatant of the Escherichia coli. Such fusion proteins are included in the present invention as derivatives of the peptide.

[0060] Instead of phagemids, various vectors, circular or non-circular, preferably viral vectors, may be used. The nucleotide sequences of the present invention contained in the vector can be expressed by any method known to those skilled in the art. The peptide encoded by the base sequence of the vector is then transfected into a cell or virus. or expressing or presenting (synonymous with display) the protein on a globulin-like particle, or culturing the cell. It can be produced in the supernatant.

[0061] The vector (recombinant vector) of the present invention can be prepared by a method well known to those skilled in the art, such as genetic recombination. It can be manufactured.

[0062] The vector of the present invention can be in an isolated form (lyophilized preparation, solution, etc.), bound to other molecules (solid-phase, etc.), or introduced into cells (this a physical assembly (including a recombinant cell of the invention), other vectors, etc. Examples of such examples include, but are not limited to, the following: Instead, any form suitable for the purpose of use, storage, etc. can be selected.

[0063] 4.Cells In one aspect, the present invention provides a recombinant cell (hereinafter simply referred to as "cell"). To provide.

[0064] The cells of the present invention contain nucleotides corresponding to the peptides of the present invention and express the peptides. Host cells for such cells include eukaryotic cells (established cell lines, primary culture cells, etc.). There is no particular limitation on the type of cells, and they may be either prokaryotic cells or subcultured cells. .

[0065] Examples of prokaryotic cells include Escherichia coli, Bacillus subtilis, Examples of bacterial cells include those of Bacillus subtilis. It is not limited to:

[0066] Examples of eukaryotic cells include animal cells, insect cells, yeast, fungi, etc. As an example of animal cells, there are COS cells, which are monkey cells (Glutzman, Cell, Vol. 23, Published in 1981, pp. 175-182: Gluzman, Y., Cell (198 1), vol.23, pp175-182: American Type Culture Collection ATCC CRL-1650), mouse fibroblast cells NIH3T3 (American Kang Type Culture Collection No. ATCC CRL-1658), Chai Chinese hamster ovary cells CHO cells: American Type Culture Collection (ATCC) CCL -61), a dihydrofolate reductase-deficient CHO cell line (Ulraop and Chasin, Proc. Proceedings of the National Academy of Sciences, USA, Vol. 77 , published in 1980, pp. 4126-4220: Urlaub, G. and Chas. in, LA, Proc. Natl. Acad. Sci. USA (1 980), vol.77, pp4126-4220), etc. However, the present invention is not limited to these.

[0067] The cells of the present invention can be produced by introducing the nucleotide or vector of the present invention into a host cell. However, it is preferable to prepare the vector of the present invention by transfection, transformation, etc. They can be prepared by introducing them into host cells by transformation, transduction, or the like.

[0068] Vectors that can be applied to prepare the cells of the present invention include, for example, vectors that are compatible with such prokaryotic cells. A replicon or origin of replication from the species from which it is derived, along with regulatory sequences, transcription start site, and (translation) initiation site. Plasmids and cosmids containing one or more base sequences selected from the group consisting of a stop codon and a termination codon. Examples of such vectors include, but are not limited to, phagemids, etc. Such a nucleotide or vector is then introduced into a cell. The vector may contain a base sequence that can confer selectability of a phenotype. Alternatively, the nucleotides can be introduced into host cells and the resulting cells can be cultured to produce the compounds of the present invention. The peptide can be expressed.

[0069] Host cells suitable for post-translational modification of the peptides of the invention may also be used as cells of the invention. The cells of the present invention to which such host cells are applied can be used, for example, to express the peptide-inducing gene of the present invention. It can be used in (some embodiments of) a method for preparing the body.

[0070] The cells of the present invention can be in an isolated form (frozen sample, freeze-dried sample, etc.). The nucleotides of the present invention may be in the form of a preparation, a solution, etc., a form bound to other molecules (a solid phase form, etc.), Cells into which a gene or vector has been introduced (included in the cells of the present invention), Cells (included in the cells of the present invention) that express or display (synonymous with display) on their surface , other cells, etc. (nucleotide library and peptide library of the present invention) Examples of such examples include, but are not limited to, the following: Instead, any form suitable for the purpose of use, storage, etc. can be selected.

[0071] 5. Peptide Production Method In another aspect, the present invention provides a method for producing the peptide of the present invention.

[0072] As described above, the peptide of the present invention can be produced by chemical synthesis, genetic recombination, in vitro translation, etc. They can be prepared by methods well known to those skilled in the art for producing peptides and proteins. In addition, the identification method of the present invention can be used to recover (select, concentrate, isolate) the desired protein from the library of the present invention. Peptides containing hydroxybenzoates (including hydroxybenzoates) can also be prepared by these methods.

[0073] In one embodiment, the method for producing the peptide of the present invention comprises the following steps (1-1) and (1- 2) comprising: (1-1) A vector containing a nucleotide corresponding to the peptide of the present invention and expressing the peptide, etc. Culturing cells (cells of the invention); and (1-2) recovering the peptide from the culture.

[0074] In another embodiment, the method for producing a peptide of the present invention comprises the following steps (2-1): (2-2) (2-1) determining the amino acid sequence of the peptide of the present invention that binds to a target molecule; and , (2-2) A peptide consisting of the amino acid sequence is prepared by chemical synthesis or genetic recombination. The process.

[0075] Furthermore, in another embodiment, the method for producing the peptide of the present invention comprises the following steps (3-1): and (3-2): (3-1) preparing mRNA corresponding to the peptide of the present invention; and (3-2) Using the mRNA obtained in (3-1) as a template, Preparing the peptide.

[0076] In addition, these manufacturing methods may be appropriately combined with the identification method of the present invention as a preliminary step. That is, first, the steps included in the identification method of the present invention are carried out, and then the steps of the present invention are carried out. The method for producing the peptide of the present invention can be carried out by carrying out the steps included in the method for producing the peptide of the present invention. The present invention also encompasses a method further comprising (each step of) the identification method of the present invention, such as:

[0077] The method for producing such a peptide of the present invention comprises, for example, the following steps (4-1) to (4-3) ) comprising: (4-1) Contacting a peptide contained in the peptide library of the present invention with a target molecule the process of (4-2) recovering the peptide that binds to the target molecule; and (4-3) The recovered peptides can be synthesized by chemical synthesis, genetic recombination, or in vitro translation. A process for preparing the same.

[0078] Similarly, in these manufacturing methods, the determination method of the present invention may be appropriately combined as a preliminary step. That is, first, the steps included in the determination method of the present invention are carried out, and then the steps of the present invention are carried out. The steps included in the production method of the present invention can be carried out. The present invention also encompasses a method that further comprises (each step of) such a determination method of the present invention. do.

[0079] The method for producing such a peptide of the present invention comprises, for example, the following steps (5-1) to (5- 3) comprising: (5-1) contacting the test peptide of the present invention with a target molecule; (5-2) If the test peptide binds to the target molecule, the peptide is determined to be positive. and (5-3) If the test peptide is determined to be positive in (5-2), Preparing the peptide by chemical synthesis, genetic recombination or in vitro translation.

[0080] Furthermore, in another embodiment, the method for producing the peptide of the present invention comprises: The target molecule is other than trypsin and / or acrosin, preferably other than trypsin. It binds to a target molecule and activates or promotes some or all of the biological activity of the target molecule. and identifying peptides that promote, inhibit, inactivate or suppress the activity of the protein. Such a production method is, for example, the following (6-1) to (6-3) or (7-1) to (7-3) ) process: (6-1) Contacting a peptide contained in the peptide library of the present invention with the target molecule The process of making (6-2) recovering the peptide that binds to the target molecule; and (6-3) whether the peptide activates or promotes the biological activity of the target molecule; or determining the peptide as positive if it agonizes the target molecule. (7-1) Contacting a peptide contained in the peptide library of the present invention with the target molecule The process of making (7-2) recovering the peptide that binds to the target molecule; and (7-3) The peptide inhibits, inactivates, or suppresses the biological activity of the target molecule. or antagonizes the target molecule, the peptide is considered positive. A process of determining.

[0081] In another aspect, the production method of the present invention is (6-1) to (6-3) or (7 Instead of (7-1) to (7-3), for example, the following (8-1) and (8-2) or (9 -1) or (9-2): (8-1) A test peptide and a compound other than trypsin and / or acrosin, preferably trypsin contacting the antibody with a target molecule other than the antibody; and (8-2) whether the peptide activates or promotes the biological activity of the target molecule; or determining the peptide as positive if it agonizes the target molecule. (9-1) A test peptide and a compound other than trypsin and / or acrosin, preferably trypsin contacting the antibody with a target molecule other than the antibody; and (9-2) The peptide inhibits, inactivates, or suppresses the biological activity of the target molecule. or antagonizes the target molecule, the peptide is considered positive. A process of determining.

[0082] In steps (6-1) to (6-3), or (8-1) and (8-2), etc., By combining steps similar to 3) or (5-3), etc., it is possible to obtain a target molecule-containing organism. Peptides that activate or promote activity or stimulate target molecules (ago Similarly, (7-1) to (7-2) can be produced. -3), or (9-1) and (9-2), etc., followed by (4-3) or (5-3) By combining similar processes, the biological activity of the target molecule can be inhibited or inactivated. or peptides that inhibit the activity of target molecules, or peptides that antagonize target molecules tic peptide) can be produced.

[0083] The present invention also provides a method for producing a peptide derivative (peptide derivative). The peptide derivatives can be prepared by, for example, converting the peptide contained in the derivative into a peptide derivative by the above-mentioned method (peptide production method). After preparation by the above method, the prepared protein is subjected to chemical reactions, biochemical reactions, post-translational modifications, etc. It can be made from, but is not limited to,

[0084] The peptide prepared in the above (2-2), (3-2), (4-3) or (5-3) Instead of the amino acid sequence of the peptide (X), one or more amino acids are selected from the amino acid sequence of the peptide (X). A peptide (X') having an amino acid sequence in which a partial amino acid sequence is deleted is prepared. The method for producing peptide (X') is also included in the present invention. The prepared peptide (X') preferably binds to a target molecule. The amino acid sequence of the original peptide is One or more amino acids or partial amino acid sequences that are not essential for binding to the molecule may be deleted. stomach.

[0085] The peptide derivatives of the present invention can be produced, for example, by the above-mentioned (1-1) and (1- 2), (2-1) and (2-2), (3-1) and (3-2), (4-1) to (4 -3), (5-1) to (5-3), (6-1) to (6-3), (7-1) to (7- 3), (8-1) and (8-2), or (9-1) and (9-2), etc. In addition, a step of preparing a peptide derivative of the present invention using the peptide of the present invention as a raw material (hereinafter referred to as (2-2), (3-2), and (4-3) are also included in the above-mentioned methods (hereinafter referred to as "derivative preparation steps"). -3) and (5-3), a step of preparing a peptide derivative instead of the peptide. a method for incorporating peptide derivatives into a peptide library in advance; Examples of methods include using a test peptide derivative instead of the test peptide. However, the present invention is not limited to these.

[0086] The above (2-2), (3-2), and (4-) can be included in the methods for producing the peptide derivatives of the present invention. Instead of the peptide derivative prepared in 3) or (5-3), One or more amino acids or partial amino acid sequences are selected from the amino acid sequence of the target (Y). It is also possible to prepare a peptide derivative (Y') having a deleted amino acid sequence. The present invention also encompasses a method for producing the peptide derivative (Y'). The peptide derivative (Y') preferably binds to a target molecule. The amino acid sequence of the original peptide derivative is included in the amino acid sequence of the original peptide derivative. One or more amino acids or partial amino acid sequences that are not essential for binding to the target molecule are deleted. In addition, cells having the desired post-translational modification ability may be used in the production of the peptide of the present invention. By using the peptide of the present invention in a manner similar to that described above, the peptide of the present invention can be obtained as a peptide having the desired post-translational modification. Derivatives can also be prepared. In such cases, for example, cells with the desired post-translational modification ability can be used. as the cells in the above steps (1-1) and (1-2), or (2-2), Cells (or host cells) used in genetic recombination in (4-3) and (5-3) By using each of these, a peptide derivative with the desired post-translational modification can be prepared. However, the translation of peptides (as an embodiment of the method for producing peptide derivatives) of the present invention can be The method for preparing post-translationally modified forms is not limited thereto.

[0087] 6. Library The present invention provides a library.

[0088] In the present invention, a "library" is a physical collection of similar, but non-identical, molecules. Such a collection may, for example, coexist in one container, but may be different. as groups or as individual molecules at different locations on a container or solid support. The libraries may be physically separated. Multiple libraries may be included in the same collection. Good too.

[0089] The libraries of the invention may contain non-identical peptides and / or nucleotides of the invention. The physical assembly of the vectors is not limited to any particular method, but may be, for example, a method using phage display. Libraries, Ribosome Display Libraries, Nucleic Acid Display Libraries Examples include rally.

[0090] "Phage display" refers to the use of filamentous phages. A foreign peptide or protein is linked to a coat protein such as ge. The resulting fusion protein is expressed or displayed (synonymous with display) on a phage-like particle. It also refers to the technology (method and means) that utilizes such technology. The recovery (including selection, enrichment, and isolation) of nucleotides corresponding to peptides or proteins is also called "Farmer." Phage display libraries are included in the meaning of "phage display." This is one embodiment of the library of the present invention used in such a technique.

[0091] "Ribosome display" refers to the display of ribosomes formed during the translation reaction in in vitro translation. As a complex comprising three components: mRNA, ribosome, and peptide or protein. A technique for expressing or presenting (synonymous with display) a peptide or protein in the form of Here, the peptide or protein is a peptide or protein derived from the mRNA. Furthermore, the nucleic acid corresponding to the peptide or protein can be obtained by such a technique. The recovery of nucleotides (including selection, enrichment, and isolation) is also included in the meaning of "ribosome display." Ribosome display libraries are used in such techniques. This is another embodiment of the library of the present invention.

[0092] "Nucleic acid display" refers to a method of displaying a nucleotide (synonymous with nucleic acid) and a corresponding nucleic acid. The peptide or protein is in the form of a complex comprising the peptide or protein. It refers to the technology (method and means) for expressing or presenting (synonymous with display). (Keefe and Szostak, Nature, Vol. 410, pp. 715-718, 2001) Published by: Keefe, AD and Szostak, JW, Nature, vol.410 (2001), pp715-718). In addition, The recovery (including selection, enrichment, and isolation) of nucleotides corresponding to the peptide or protein is also referred to as " The term "nucleic acid display" includes within its meaning. Another embodiment of the library of the present invention is used in the art.

[0093] Examples of nucleic acid display include mRNA display, However, it is not limited to this. (Yamaguchi, J. et al., Nu Cleic Acids Research, vol.37, No.16 e108 , pp1-13 (2009)) mRNA display is mediated by mRNA and its translation product, a peptide or protein. The peptide or protein is presented in the form of a complex consisting of two or more peptides linked together (displayed). (Keefe and Szostak, Nature, Vol. 410, p. 71) Pages 5-718, published 2001: Keefe, AD and Szostak, JW, Nature, vol.410 (2001), pp715-718) .

[0094] In the present invention, the physical properties of the peptides and / or nucleotides of the present invention that are not identical physical aggregation of cells, including biological aggregation, microorganisms (viruses, phages, phage-like molecules, etc.) a physical assembly of particles, whether naturally occurring or artificially created, Physical assembly of vectors (including phagemids, cosmids, plasmids, etc.) and the physical assembly of those fragments, and the chemical and / or biological modifications thereof. A physical collection is also included within the meaning of "library."

[0095] As described above, in the present invention, when designing a base sequence encoding an amino acid sequence, can use one or more codons corresponding to each amino acid. A base sequence encoding a single amino acid sequence of a peptide or protein may be composed of multiple bases. In selecting such codons, the gene corresponding to the peptide may have variations. The codon usage of the cell (host cell) into which the nucleotide containing the base sequence is introduced is determined. Codons can be selected appropriately depending on the codon usage, and multiple codons can be used. The frequency or ratio can be appropriately adjusted. In the library, the nucleotides of the base sequence encoding a single amino acid sequence are That is, the nucleotide library of the present invention may have certain variations. The physical assembly of nucleotides containing the base sequence that codes for the amino acid sequence of a peptide The physical collection of nucleotides corresponding to such a particular peptide may be These themselves can form a nucleotide library.

[0096] The libraries of the present invention contain a plurality of similar, but not identical, molecules. The number of similar molecules contained in a library is called the "library diversity." For example, the diversity of a library of 100 similar molecules is 10 2 In the present invention, The diversity of the library is not particularly limited, but the higher the value, the more preferable. stomach.

[0097] The present invention relates to a method for identifying a peptide and a method for producing a peptide comprising the steps of the method for identifying a peptide. The diversity of the resulting peptide library is 1 × 10 5 That's it, 2 x 10 5 That's it, 5 x 10 5 Below Top, 1×10 6 That's it, 2 x 10 6 That's it, 5 x 10 6 That's it, 1 x 10 7 That's it, 2 x 10 7 Below Top, 5×10 7 That's it, 1 x 10 8 That's it, 2 x 10 8 That's it, 5 x 10 8 That's it, 1 x 10 9 Below Top, 2×10 9 That's it, 5 x 10 9 That's it, 1 x 10 10 That's it, 2 x 10 10 That's it, 5 x 10 10 That's it, 1 x 10 11 That's it, 2 x 10 11 That's it, 5 x 10 11 That's it, 1 x 10 12 End , 2 × 10 12 That's it, 5 x 10 12 That's it, 1 x 10 13 That's it, 2 x 10 13 That's it, 5 x 1 0 13 That's it, 1 x 10 14 That's it, 2 x 10 14 That's it, 5 x 10 14 That's it, 1 x 10 15 Below Top, 2×10 15 That's it, 5 x 10 15 That's it, 1 x 10 16 That's it, 2 x 10 16 That's it, 5x 10 16 or more, or 1×1017 The diversity of this library is The value is not limited to this value, and may be a theoretical value.

[0098] 7. Identification Method The present invention provides a method for identifying peptides and / or peptide derivatives that bind to a target molecule. The identification method of the present invention can be carried out by, for example, the above-mentioned steps (4-1), (4-2), (6- 1) and (6-2), (7-1) and (7-2), etc. may be included, but Not limited.

[0099] (1)Target molecule In the present invention, the term "target molecule" refers to a human or non-human target molecule to which the peptide of the present invention binds. It means a substance present in an individual animal or an exogenous substance that can be taken into the living body. The target molecule is preferably trypsin and / or trypsin, which are endogenous targets of SPINK2. A molecule other than acrosin, more preferably other than trypsin, and even more preferably of human origin. Even more preferably, the molecule is derived from a human other than trypsin. may be directly or indirectly involved in the onset or aggravation of a disease to which a person may be susceptible, or Endogenous or exogenous enzymes, receptors, or receptors that correlate or inversely correlate with the disease Ligands of the body, humoral factors such as cytokines, other biopolymers, signal transduction substances, Bacteria, pathogens, toxins, or substances derived from any one or more of these, e.g. For example, their fragments, degradation products, metabolic products, processed products, etc. (hereinafter referred to as "disease-related target molecules"). The target molecules of the present invention may be minerals, polymers, plastics, synthetic low molecular weight compounds, etc. The substance may be a non-natural substance.

[0100] In some embodiments of the present invention, preferred target molecules are those other than trypsin and / or acrosin. , more preferably a protease other than trypsin, and even more preferably a serine protease. Even more preferably, it is an endo-type serine protease, and An example of a phosphoprotease is chymotrypsin. When purifying and isolating specific protein components from cells, etc., the fraction containing such components contains the proteins. Addition of protease inhibitors can reduce the degradation of such components, and Proteinase inhibitors are useful in the production of a variety of proteins, both recombinant and non-recombinant. Furthermore, these proteases are preferably disease-related target molecules.

[0101] The target molecule of the present invention is a peptide that binds to the target molecule from the peptide library of the present invention. When selecting peptides that stimulate the target molecule or peptides that antagonize the target molecule, When determining the binding activity or stimulatory or antagonistic activity of a test peptide to a target molecule, Such target molecules may be full-length molecules or fragments thereof, or any amino acid sequence. It may also be a derivative to which a peptide, protein, sugar chain, polymer, carrier, etc. is added. Such target molecules may be immobilized.

[0102] (2) Preparation of target molecules The target molecule of the present invention can be isolated or purified from diseased tissues or cells, or can be administered to a subject by injecting the target molecule into a tissue or cell. It can be used in a form in which all or part of it is bound to or contained in cells, etc. The target molecules of the invention may be peptides or proteins, which may be synthesized chemically, recombinantly, or in vitro translated. The product thus obtained can be prepared by a method well known to those skilled in the art. If necessary, derivatives such as those described above may be prepared from the target molecule thus obtained.

[0103] In the present invention, peptides or proteins are prepared by in vitro translation, i.e., Nucleotides of DNA, cDNA, etc. corresponding to the peptide or protein, or the nucleotides A vector containing the above is placed in a solution containing enzymes, substrates, energy substances, etc. necessary for transcription and translation. A method for synthesizing a desired peptide or protein in vitro by incubating at 47°C. Recombination, i.e., the introduction of the nucleotide or vector into a prokaryotic or eukaryotic cell (host The resulting recombinant cells are cultured, and the desired peptide or It can be prepared by a protein recovery method, chemical synthesis, or the like.

[0104] When the target molecule is a protein or a domain thereof present in the cell membrane, such a protein or is a fusion protein consisting of the extracellular region of the domain linked to the constant region of immunoglobulin (Ig). The molecule is expressed in an appropriate host-vector system to produce a secreted protein. can also be prepared.

[0105] The nucleotide sequence corresponding to the target molecule can be obtained, for example, by expression cloning. Expression cloning methods can be used to express peptides or proteins, but are not limited to these. constructing an expression library of cDNA containing a base sequence encoding the amino acid sequence of the white matter; Using such a cDNA library as a template, the entire length or a part of the cDNA is specifically amplified. Using the primers, polymerase chain reaction (hereinafter referred to as "PCR": Saiki et al., Saiki et al. 239, pp. 487-489, 1988: Saiki, RK, et al., Science (1988), vol.239, pp487- 489) to clone the cDNA corresponding to the peptide or protein. This is a method.

[0106] Kits and reagents applicable to in vitro translation include, for example, those from Roche Diagnostics. Examples include the Rapid Translation System (RTS) manufactured by Tix. do.

[0107] As a host cell for genetic recombination, it is used as a host cell for preparing the cell of the present invention. Any prokaryotic or eukaryotic cell can be selected as appropriate.

[0108] Recombinant cells obtained through genetic recombination (nucleotides or vectors introduced The cells can be cultured according to methods well known to those skilled in the art, and the cells can be cultured or grown in the culture. The desired peptide or protein can be produced intracellularly.

[0109] The medium used for such culture can be appropriately selected from those commonly used depending on the host cell. When the host cell is E. coli, for example, ampicillin can be added to the LB medium as needed. The culture can be carried out by adding antibiotics such as lactic acid bacteria or IPTG.

[0110] The desired peptide or protein produced inside or outside the recombinant cells by such cultivation is The physical, chemical and / or biological properties of the The compound can be purified and isolated by combining the compound with other compounds.

[0111] Such fractionation techniques include, for example, salting out, treatment with protein precipitants, dialysis, ultrafiltration, Molecular sieve (gel filtration) chromatography, adsorption chromatography, ion exchange chromatography chromatography, affinity chromatography, partition chromatography, hydrophobic Examples of suitable methods include, but are not limited to, chromatography.

[0112] In addition, a moiety useful for purification may be linked or added to a peptide or protein in advance. By doing so, the desired peptide or protein can be efficiently purified. By linking a six-residue histidine tag in advance, nickel affinity The desired peptide or protein can be efficiently purified by affinity chromatography. In addition, by linking the Fc region of IgG in advance, Protein A affinity can be Efficient purification of desired peptides or proteins by community chromatography It can be done

[0113] (3) Contact of the target molecule with the peptide and / or peptide derivative The identification method of the present invention comprises contacting a peptide and / or a derivative thereof with a target molecule. wherein the peptide and / or its derivative is added to a peptide library. That is, the identification method of the present invention can be used to identify a peptide contained in a peptide library. The method may include a step of contacting the target molecule with a peptide and / or a derivative thereof.

[0114] In the present invention, "contacting" refers to bringing two or more substances into contact with each other. This means that two or more objects are brought close enough to interact with each other. are, for example, covalent bonds, coordinate bonds, metal-metal bonds, ionic bonds, metallic bonds, and hydrogen bonds. , van der Waals bonds, etc. (hereinafter referred to as "chemical bonds"), bonds due to Coulomb forces, Electrostatic interactions such as ionic interactions, hydrogen bonds, dipole-dipole interactions, and van der Waals forces Interactions based on molecular force (hereinafter referred to as "intermolecular forces"), other interactions, charge transfer interactions These include interactions, transannular interactions, hydrophobic interactions, and associations between peptides and biomolecules. However, the present invention is not limited to these. The term "test substance" is not particularly limited as long as it includes a target molecule and a test substance. The substance is not particularly limited as long as it binds to the target molecule. The peptide of the present invention, a derivative of the peptide, a carrier on which any of them is immobilized, Cells, virus particles or viruses in which either is expressed or displayed (synonymous with display) Examples of such specimens include virus-like particles (including phages and phagemids). Quality is measured by phage display, ribosome display, nucleic acid display, etc. on the surface of a eukaryotic or prokaryotic cell, on a virus or virus-like particle, or on a Expressed or presented (synonymous with display) in a form linked to a polypeptide or nucleic acid That's fine.

[0115] (4) Selection The identification methods of the present invention identify peptides and / or peptide derivatives, preferably those with desired properties. The step of selecting peptides and / or peptide derivatives that bind to the target molecule is included.

[0116] In the present invention, "binding" means that two or more substances are bound together under certain conditions. To the extent that interactions (described elsewhere in this invention) can occur between them, It means to be in close proximity or association.

[0117] In the present invention, a target molecule is contacted with a test substance under certain conditions, and then the target Analyte substances that are non-specifically adsorbed to the molecule and analyte substances that are not bound to or adsorbed to the target molecule If the test substance is still present in the fraction after removal of the test substance from the fraction containing the test substance, A given analyte can be considered to "bind" to the target molecule.

[0118] When only nonspecific adsorption occurs between two or more substances, It can be understood that no "bond" occurs between the substances. When the substances are brought into contact, the interactions (described elsewhere in this invention) between them are not If they are not in close proximity or association with each other to the extent that this can occur, It can be understood that no "bond" occurs between the substances.

[0119] The "binding" of antibodies and antigens can be measured by flow cytometry, etc. Fluorescent antibody techniques (direct and indirect), radioimmunoassays, and enzyme immunoassays are performed. (homogeneous and heterogeneous methods), ELISA, ELISPOT, etc. are widely used. In the method, the test antibody and antigen are combined with the peptide or derivative thereof of the present invention and the target molecule. In other words, the measurement of the "binding" between an antibody and an antigen is similar to the measurement of the binding between an antibody and an antigen, and the measurement of the binding between an antibody and an antigen is similar to the measurement of the binding between an antibody and an antigen, but the measurement of the binding between an antibody and an antigen is similar to the measurement of the binding between an antibody and an antigen. The presence or absence of "binding" of target molecules can be measured.

[0120] The presence or absence of "binding" can be determined by measuring an index of binding activity or affinity. Indicators of binding affinity include dissociation constants, binding constants, etc. do.

[0121] Regarding the chemical dissociation of molecule A and substance B bound to A in the chemical equilibrium state shown below, And,

[0122] [ka]

[0123] The dissociation constant (Kd) is calculated using the following formula: You can: Kd = [A][B] / [AB] Here, [A], [B], and [AB] are the concentrations of A, B, and AB (aggregates), respectively. Kd means the ratio of dissociated A and B to undissociated AB. The reciprocal of Kd is the binding constant (Ka).

[0124] The "dissociation constant" used in the present invention ) is primarily a method for the synthesis of peptides and / or peptide derivatives for binding to a target molecule. It means the equilibrium dissociation constant.

[0125] In the present invention, the dissociation constant is the dissociation constant of a dissociated substance (peptide, peptide derivative, target molecule, etc.) ) and undissociated substances (peptide and / or peptide derivative-target molecule complexes The dissociation constant can be calculated by measuring the concentration of the The method is not particularly limited as long as it is a method well known to those skilled in the art. For example, Examples of the method include a method using Sumon resonance and an isothermal titration calorimetry method.

[0126] In the method using surface plasmon resonance, a target molecule, a peptide that binds to the target molecule, and The interaction of the peptides and / or derivatives thereof can be measured and calculated as follows: A series of binding and dissociation reactions are detected by surface plasmon resonance at peptide concentrations; Analyze the series of binding and dissociation reactions obtained; calculate the dissociation constant from the various rate constants obtained .

[0127] As a surface plasmon resonance measurement-calculation system, for example, Biacore e) Systems (GE Healthcare), etc., but are not limited to these. The procedure for using the Biacore system is as follows: Immobilization of target molecules on the sensor chip of the core system by amine coupling; The target molecule is contacted with the peptide at a peptide concentration of Detected by plasmon resonance; a series of binding and dissociation reactions are shown on the horizontal axis as time and on the vertical axis as binding. The amount (RU) is plotted as a sensorgram; plotted at multiple peptide concentrations. From the sensorgrams, BIAevaluation software (GE Healthcare are) and fitted to a 1:1 Langmuir model. Calculate the parameters; calculate the dissociation constant from various rate parameters.

[0128] In the isothermal titration calorimetry method, the target molecule and its binding peptide and / or or its derivatives can be measured and calculated as follows: Add the peptide solution to the solution (or vice versa); measure the heat generated by the interaction and Draw the binding isotherm; from the binding isotherm, calculate the dissociation constant (K D), bond ratio of the reaction (N), enthalpy The change in energy (ΔH) and entropy change (ΔS) are obtained.

[0129] Direct measurement of minute heat changes (exothermic or endothermic changes) caused by intermolecular interactions Examples of systems that can be used include the MicroCal system (GE Healthcare) Examples include, but are not limited to, MicroCal The procedure when using the system is as follows: A sample cell is placed in a constant temperature chamber. The solution is titrated and stirred; the intermolecular interactions result in the generation of heat proportional to the amount of binding. Absorption occurs, changing the solution temperature in the sample cell; cell feedback network The CFB senses the temperature difference (ΔT) with the reference cell; Heat the reference cell or sample cell; the temperature required to maintain ΔT = 0 By measuring the back-feedback power, the amount of heat generated or absorbed by the interaction can be obtained; The vertical axis represents the molar ratio of the target molecule to the peptide, and the horizontal axis represents the dissociation constant calculated from the binding isotherm. .

[0130] In the identification method and / or determination method of the present invention (described later), for example, and 100 μM or less, 50 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 2 μM Below, 1 μM or less, 500 nM (0.5 μM) or less, 200 nM or less, 100 nm or less, 50nM or less, 20nM or less, 10nM or less, 5nM or less, 2nM or less, 1nM or less, 5 00pM (0.5nM) or less, 200pM or less, 100pM or less, 50pM or less, 20p The dissociation constant is ≤10 pM, ≤5 pM, ≤2 pM, or ≤1 pM. A test substance that binds to the target molecule, i.e., can be determined to be positive, but has a dissociation constant The reference value and the criteria for determining the presence or absence of "binding" are not limited to these.

[0131] In the identification method of the present invention, the "selection" step is a step of recovering test substances that bind to the target molecule. The product contains or is concentrated in the form of a test substance that binds to the target molecule. If condensed, the product consists solely of material that binds to the target molecule; and The target molecule may contain a substance that does not bind to the target molecule. The test substance that binds to the target molecule contained or concentrated in the product is a single substance. It may also be a mixture of two or more kinds.

[0132] The selection step, i.e., the recovery step, in the identification method of the present invention is a step of recovering a substance that binds to a target molecule. Such a process refers to a process for recovering a fraction containing or enriched in the There are no particular limitations on the fractionation-purification method as long as it is well known to those skilled in the art. The substances bound to the target molecule, the substances not bound to the target molecule, and the substances non-specific to the target molecule a step of separating the adsorbed material from the target molecule (fraction containing the target molecule); The method may include a step of eluting the target molecule (separating the target molecule). It is not necessary to set a criterion for determining whether or not binding occurs, such as a dissociation constant.

[0133] In the present invention, the "selection" included in the identification method of the present invention is sometimes called "panning." In the present invention, "panning" refers to the process of subjecting the peptides and / or peptide derivatives of the present invention to a panning reaction. The conductor is contacted with a target molecule, and a peptide and / or peptide derivative that binds to the target molecule is then added. This means recovering (including selection, concentration, and isolation) conductors.

[0134] Panning can be performed by methods well known to those skilled in the art, such as solid-phase panning, liquid panning, etc. Examples of the method include, but are not limited to, solid-phase panning. For example, the target molecule is immobilized on a solid phase, and then the target molecule is contacted with a peptide contained in a liquid phase. Then, peptides that did not bind to the target molecule and peptides that bound non-specifically were removed. After removal, the peptides bound to the target molecule are selectively removed from the solid phase (the target molecule bound to the solid phase). By separating the peptides from the target peptides, peptides with the desired binding activity can be selected. The procedure of the solid-phase panning method is not limited to this. In the liquid-phase panning method, for example, The peptide is contacted with the target molecule in a solution, and then the peptide that did not bind to the target molecule is removed. After removing non-specifically bound peptides, peptides that bound to the target molecule were selected. and selectively separating the peptide from the target to select a peptide having the desired binding activity. However, the procedure of the liquid phase panning method is not limited to this.

[0135] In the identification method of the present invention, Using a library in which the genotype (synonymous with genetic trait) is linked By this, peptides that bind to target molecules (including "peptides contained in peptide derivatives") are obtained. The nucleotides corresponding to the nucleotides (including the nucleotides of the nucleotide sequence) are efficiently selected, and thus the peptides are efficiently prepared. It becomes possible to distinguish between phenotypes and their corresponding genotypes (hereinafter simply referred to as "phenotypes and genotypes"). The link may be either direct or indirect.

[0136] A phenotype and a genotype are "directly linked" when the behavior of the phenotype and the genotype are consistent. This means that there is a certain distance between the phenotype and the genotype, such as the presence of other parts. However, if the behavior of both is consistent, it is included in the meaning of "directly linked." That is, it is not essential that the two be physically adjacent to each other.

[0137] In the present invention, the "matched behavior" of the phenotype and genotype means that the peptides of the present invention, Nucleotides, vectors, cells, production methods, identification methods, determination methods, peptide libraries The behavior of both is consistent in terms of the nucleotide library, composition, reagent, etc. In these aspects, it means that over time, temporarily, up to a certain point, if Or, after a certain point in time, due to internal factors, external factors, a combination of these, or other factors, Even if the "matching behavior" is lost in whole or in part, the meaning of "matching behavior" remains the same. Included.

[0138] An example of a direct link between phenotype and genotype is the ribosome display gene. nucleic acid display library, nucleic acid corresponding to the peptide of the present invention Peptides and / or peptides characterized in that they are directly or indirectly linked to and derivatives thereof, peptide live compounds comprising such peptides and / or derivatives thereof. Examples include rally.

[0139] When phenotype and genotype are "indirectly linked," the behavior of the two does not necessarily coincide. Or, the two are not necessarily "directly linked," but rather a specific phenotype can be linked to the phenotype. This means that the genotype corresponding to the phenotype can be accessed. Examples of these include phage display libraries, expression cloning, and Examples of the cDNA library used in the isn't it.

[0140] Each clone contained in a phage display library or cDNA library In this study, peptides or their derivatives as phenotypes and corresponding genotypes Although the behavior of the nucleotides in the library is not necessarily consistent, The target molecule is contacted with the amide and / or its derivative, and the target molecule is either not bound to the amide or not bound to the amide. After removing phage-like particles that were nonspecifically adsorbed to the target molecule, the phage-like particles that bound to the target molecule were removed. By going through a process such as selectively eluting the phage-like particles, they bind to the target molecule. Selection of peptides and / or their derivatives (expressed or displayed on phage-like particles) and the corresponding genotypes, i.e., such peptides or their derivatives. The nucleotides corresponding to the peptides contained in the protein are purified and isolated, and their base sequences are determined. This is an advantage in that it allows access to the corresponding genotype from the phenotype. The case is limited to cases where phenotype and genotype are "indirectly linked," such as phage display. This is not a fixed term, and can also be enjoyed when there is a "direct link."

[0141] The steps involved in the identification method of the present invention can be repeated two or more times. The peptides and / or peptide derivatives recovered in the selection step are used to generate peptides again. A library of peptides is constructed and subjected to contact and selection processes to identify peptides that bind to the target molecule. This makes it possible to more highly enrich the peptides and / or derivatives thereof that bind to the peptides. By further repeating these procedures, peptides and / or peptides that bind to the target molecule can be obtained. The derivatives of β-glucan are more highly enriched, ultimately increasing the efficiency of their isolation. Furthermore, peptides and / or derivatives thereof that bind to target molecules are more highly enriched. This allows for the isolation of binders with higher affinity.

[0142] Furthermore, a high degree of enrichment of peptides and / or derivatives thereof that bind to target molecules can be achieved by the present invention. In the steps included in the method for identifying a target-binding peptide and / or a derivative thereof This can also be achieved by more strongly separating specific and non-specific binding. As an effective separation method, for example, the number of steps for removing non-specifically bound peptides and the like may be increased. or by using stronger reagents (such as detergents) to remove non-specifically bound peptides. Examples of such measures include changing the system to one that is more powerful.

[0143] The peptides of the present invention or derivatives thereof may be in the form of a monomer, a homo- or hetero-dimer, a trimer, or the like. , tetramer, pentamer, hexamer, heptamer, octamer, or composed of nine or more monomers The protein may be in any form, including a polymer.

[0144] The peptide or its derivative of the present invention that binds to one target molecule or one target site The number of molecules may be 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more. The peptide or its derivative may be in the form of a monomer, homo- or hetero-dimer, trimer, tetramer, pentamer, a dimer, hexamer, heptamer, octamer, or a multimer consisting of nine or more monomers. It may bind to the target molecule in either form.

[0145] The number of molecules of the target molecule that binds to one molecule of the peptide of the present invention or its derivative The number of target sites may be 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more.

[0146] The present invention also provides a method for activating or promoting a part or all of the biological activity of a target molecule. or methods for identifying peptides or peptide derivatives that inhibit, inactivate or suppress and the preparation of peptides or peptide derivatives that stimulate or antagonize target molecules. That is, the present invention provides a method for identifying activators, promoters or stimulators of target molecules. agonist, or inhibitor, inactivator, suppressor or antagonist These identification methods include, for example, the steps described above. (6-1) to (6-3), (7-1) to (7-3), etc. may be included, but the steps This is not limited to including procedures.

[0147] 8. Composition The present invention provides a composition.

[0148] The composition of the present invention includes the peptide, peptide derivative, nucleotide, vector or or cells.

[0149] The peptide of the present invention or a derivative thereof (including those displayed on the cell surface of the present invention) and detecting a target molecule to which the peptide or a derivative thereof binds. can be used for

[0150] A composition comprising a nucleotide, vector or cell of the present invention can be prepared by administering the nucleotide, vector or cell to a subject. The base sequence of the nucleotide of the present invention contained in the nucleotide, vector, or cell It can be used to prepare a peptide having the encoded amino acid sequence. In addition, such a composition can be used to detect nucleotides, vectors, cells, etc. containing the nucleotide. It can also be used for

[0151] Such compositions may optionally contain buffers, salts, metals, preservatives, surfactants, freezing or The peptides, peptide derivatives, nucleotides, and the like of the present invention can be prepared by a preparation method such as freeze-drying or freeze-drying. It may contain substances to reduce or prevent damage to vectors or cells. This can be done.

[0152] 9. Reagents The present invention provides a reagent.

[0153] The reagent of the present invention may be a peptide, a peptide derivative, a nucleotide, a vector, or a comprises a cell.

[0154] The reagent comprising the peptide of the present invention or a derivative thereof is The derivative can be used to detect the target molecule to which it binds.

[0155] A reagent comprising the nucleotide, vector or cell of the present invention is It can be used to detect nucleotides, vectors, cells, etc. containing the nucleotides.

[0156] The reagent of the present invention may be a composition. A kit containing such a reagent is also included in the reagent of the present invention.

[0157] Furthermore, the peptides, peptide derivatives, and cells on which they are presented according to the present invention can be used to treat target molecules, etc. As an element that recognizes such substances, it can be used in a biosensor for such substances. do.

[0158] 10.Judgment method The present invention also provides a method for determining whether a test substance binds to a target molecule. The method for determining brightness may include, for example, the above steps (5-1) and (5-2). But not limited to these.

[0159] The determination method of the present invention may include steps that are the same as or appropriately modified from the steps included in the identification method of the present invention. However, the test substance to be subjected to such a determination method must be a library For example, the test peptide or test peptides to be subjected to the determination method may not be included in the set. Peptide derivatives are peptides or peptide derivatives contained in a peptide library. Without limitation, a single peptide or peptide derivative isolated from other peptides. In other words, the identification method of the present invention is mainly This method is suitable as a method for selecting substances having desired properties from a physical collection of test substances. In contrast, the determination method of the present invention examines whether a specific test substance has a desired property. It is also suitable as a testing method for

[0160] In the determination method of the present invention, for example, it is determined whether a test substance binds to a target molecule. In the step, the criteria for judgment are whether or not the conditions for affinity indexes such as dissociation constants are met. In addition, the test substance can be used as a target in the same selection process as in the identification method of the present invention. If the test substance is recovered as a substance that binds to the molecule, the test substance can be determined to be positive.

[0161] The present invention also provides a method for determining whether a test substance activates or inhibits a part or all of the biological activity of a target molecule. and a method for determining whether a target gene promotes, inhibits, inactivates, or suppresses a target gene. Whether the test substance agonizes or antagonizes the target molecule The present invention also provides a method for determining whether or not a tagonization has occurred. For example, the above-mentioned steps (8-1) and (8-2), (9-1) and (9-2), etc. The present invention may include, but is not limited to, the following: [Example]

[0162] The present invention will be specifically described below with reference to examples. However, these examples are not intended to be limiting of the scope of the present invention. The scope of the present invention is not limited to the plasmids and restriction enzymes used in the examples of the present invention. The DNA modifying enzymes and the like are commercially available and can be used according to standard methods. Cloning of NA, determination of polynucleotide sequence, transformation of host cells, transformed cells The procedures used for culturing the enzyme, collecting the enzyme from the resulting culture, purifying it, etc. are also well known to those skilled in the art. It is either known or can be easily found from literature etc.

[0163] [Example 1] (1-1) Construction of a randomly mutated SPINK2 library To display the randomly mutated SPINK2 library on phage, a phage midvector was used. First, we synthesized the region containing the tTH terminator to create "fragment 1 ( pCANTAB 5E (GE) containing the lac operator The region from 2097 to 2232 (SEQ ID NO: 11) of The sequence containing the SD sequence and the phoA signal peptide (SEQ ID NO: 12) was designated "Fragment 2." ) as "fragment 3", and the phage coat protein (gene III) was The sequence derived from 5E was designated as "fragment 4," and the region containing the Ipp terminator was designated as "fragment 5." was synthesized as "Fragment 5 (SEQ ID NO: 13)". Using "Fragment 1" to "Fragment 5" as templates, The following primers 1 and 2, and KOD-plus-(TOYOBO: DNA polymerase Overlap extension PC using a PCR system (consisting of enzymes, buffers, substrates, etc.) R method ((94℃ 15 seconds, 60℃ 30 seconds, 68℃ 160 seconds) × 30 cycles) It was. Primer 1: 5'-AAAAAACGCGTCTGCGGCCGCATAGGGTAG CGAAAACCT-3' Primer 2: 5'-AAAAAGGCGCCATTCGCCATTCAGGCTGCG CAACTGTTGG-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. izard SV Gel and PCR Clean-Up System (Pro DNA was prepared using the ELISA kit (Mega). The prepared DNA fragment and pCANTAB 5E were Treat with restriction enzymes AflIII (NEB) and NarI (NEB) at 37°C for at least 1 hour. After agarose gel electrophoresis, the desired DNA fragment was excised and purified using Wizard SV G. The purified fragment was purified using the PCR Clean-Up System. was reacted overnight at 16°C using T4 DNA Ligase (NEB). The ligation reaction was carried out. The ligation solution was prepared using E. coli JM109 (TOY After adding the mixture to the 0BO solution, the mixture was left standing on ice for 30 minutes, then heat-treated at 42°C for 45 seconds, and then further cooled on ice. The mixture was left to stand for 5 minutes at RT, and then inoculated onto a 2-YT plate containing 0.1 mg / ml ampicillin. The transformation of E. coli was carried out by static cultivation overnight at ℃. The cells were cultured in Terrific Broth medium (Invi) containing 0.1 mg / ml ampicillin. The bacteria were inoculated into a 1000 ml tube containing 1000 ml of ... Plasmid DNA was collected using the iniprep Kit (Qiagen) (hereinafter referred to as The target vector is constructed by carrying out sequence analysis (called "miniprep processing"). It was confirmed that this vector was a phagemid vector, and the vector was named "phagemid vector pPR3." The phagemid vector pPR3 was constructed in (1-2). It can be used in Examples (1-3) and later instead of SPINK2(WT). Cut.

[0164] (1-2) Construction of phagemid vector pPR3_SPINK2(WT) To display the randomly mutated SPINK2 library on phage, a phage midvector was used. First, the region containing the tTH terminator was denoted as "fragment 1 (SEQ ID NO: 1)". 10) and pCANTAB 5E (GE heal) containing the lac operator. The region from 2099 to 2232 of the nucleotide sequence (nucleotide number 3 of SEQ ID NO: 11) of the The fragment 2 (from 1 to 136) was designated as "fragment 2." and the bases encoding the amino acid sequence of wild-type SPINK2, i.e., SPINK2(WT). The sequence containing the sequence (SEQ ID NO: 12) was designated "Fragment 3", a phage clone based on pCANTAB 5E. The sequence containing the nucleotide sequence of the ribosomal protein (gene III) (nucleotides 600 to 604 of SEQ ID NO: 16) The region containing the Ipp terminator is designated as "fragment 4" (from 1848 to 1848). The region was set to "Fragment 5 (SEQ ID NO: 13)". The base sequence including "Fragment 1" to "Fragment 5" (sequence The DNA having the sequence number 16) was used as a template, and the following primers 1 and 2, and KOD- plus- (TOYOBO: consisting of DNA polymerase, buffer solution, substrate, etc.) Overlap extension PCR (94°C for 15 seconds, 60°C for 30 seconds, 6 8°C for 160 seconds × 30 cycles). Primer 1': 5'-AAAAGAAGAGCGCCCAATACGCAAACCGC CTCTCC-3' Primer 2': 5'-AAAAAGAATTCATTAAACGGCAGACAAAA AAAATGTCGC-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. izard SV Gel and PCR Clean-Up System (Pro DNA was prepared using the ELISA kit (Mega). The prepared DNA fragment and pCANTAB 5E were Treat with restriction enzymes SapI (NEB) and EcoRI (NEB) at 37°C for 1 hour or more, After agarose gel electrophoresis, the desired DNA fragment was excised and purified using Wizard SV Ge The purified fragment was purified using the PCR Clean-Up System. The resulting fragment was then ligated overnight at 16°C using T4 DNA Ligase (NEB). The ligation reaction was carried out using E. coli JM109 (TOYO BO), and then left to stand on ice for 30 minutes, then heat-treated at 42°C for 45 seconds, and then further heated on ice. After leaving the mixture to stand for 5 minutes, it was inoculated onto a 2-YT plate containing 0.1 mg / ml ampicillin and then incubated at 37°C. The transformation of E. coli was carried out by statically culturing the bacteria overnight at 100°C. , Terrific Broth medium (Invitrogen) containing 0.1 mg / ml ampicillin The bacteria were inoculated onto a QIAprep 96 Turbo Microplate reader (Progen) and cultured overnight at 37°C. Plasmid DNA was collected using a niprep Kit (Qiagen) (hereafter referred to as "m The target vector was constructed by performing sequence analysis. Furthermore, the constructed vector was purified by PCR using the restriction enzyme EcoRI at 37°C for 1 hour. After Klenow treatment, the DNA was ligated with T4 DNA Ligase (NEB). The ligation reaction was carried out at 16°C for 1 hour, and the resulting product was transformed into E. coli JM109. After culturing the E. coli, miniprep was performed and the resulting DNA was sequenced. The vector constructed by this procedure was designated as "phagemid vector pPR3_SPINK2(W T)" and was used in the following examples.

[0165] (1-3) Construction of phagemid vector pPR3_stuffer_TEV Next, the TEV protein was inserted into the phagemid vector pPR3_SPINK2(WT). The TEV protease cleavage sequence and stuffer were inserted. To prepare the DNA, primers 3 and 4 were used, and an overlay using KOD-plus- - Wrap extension PCR (94℃ 15 seconds, 60℃ 30 seconds, 68℃ 10 seconds) sec) × 30 cycles). Primer 3: 5'-GCGGCCGCATAGGGTAGCGAAAACCTGTAT TTTCAGAG-3' Primer 4: 5'-GCTAAACAACTTTCAACGGTgctaccGCTC TGAAAATACAGG-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. D with the izard SV Gel and PCR Clean-Up System The resulting fragment was designated as "Fragment 6." ) as a template, PCR was performed using the following primers 5 and 6 and KOD-plus- R method ((94℃ 15 seconds, 60℃ 30 seconds, 68℃ 30 seconds) × 30 cycles) The phage coat protein (gene III) was amplified by this procedure. Primer 5: 5'-ACCGTTGAAAGTTGTTTAGCAAAACCC-3' Primer 6: 5'-CATTAAAGCCAGAATGGAAAGCGCAGTC-3 ' Furthermore, the amplified DNA fragment was used as a template, and the following primers α and β and KOD PCR using -plus- (94℃ 15 seconds, 60℃ 30 seconds, 68℃ 45 seconds) × 30 cycles). Primer α: 5'-AACACGCGTCTGCGGCCGCATAGGGTAGC -3' Primer β: 5'-AACGGATCCTCATTAAAGCCAGAATGGAA AG-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. D with the izard SV Gel and PCR Clean-Up System The prepared DNA fragment and pPR3_SPINK constructed in (1-2) were used. 2 (WT) was digested with restriction enzymes MluI (NEB) and BamHI (NEB) at 37°C for 1 hour. After the above treatment and agarose gel electrophoresis, the desired DNA fragment was excised and Purification was performed using the SV Gel and PCR Clean-Up System. The resulting fragments were reacted with T4 DNA ligase at 16°C overnight to form the ligated fragments. Ligation was performed and E. coli JM109 was transformed. After culturing the bacteria, miniprep was performed and the resulting DNA was sequenced. The constructed vector was named "phagemid vector pPR3_TEV." This was carried out according to the method described in (1-1). The constructed phagemid vectors pPR3_TEV and pcDNA3.1(+)(In Vitrogen) with restriction enzymes EcoRI (NEB) and MluI (NEB) at 37°C. After agarose gel electrophoresis, the desired DNA fragment was excised and purified by Wi Purified with Zard SV Gel and PCR Clean-Up System The purified fragment was reacted with T4 DNA ligase at 16°C overnight. A ligation reaction was carried out using the resulting DNA, and E. coli JM109 was transformed with the resulting DNA. After culturing the E. coli, miniprep was performed and the resulting DNA was sequenced. The resulting vector was named "phagemid vector pPR3_stuffer_TEV." was carried out according to the method described in (1-2).

[0166] (1-4) Construction of randomly mutated SPINK2 phagemid vector To amplify the SPINK2 region that does not require mutations, we used the amino acid sequence of human SPINK2. The base sequence encoding the above (SEQ ID NO: 14) was used as a template, and the following primers 7 and 8 were used. PCR using KOD-plus (94°C for 15 seconds, 60°C for 30 seconds, 68°C for 10 seconds) 10 seconds) × 30 cycles). Primer 7: 5'-GGTAGCGATATGAGCACCTATGC-3' Primer 8: 5'-GCACGGACCATTGCGAATA-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. D with the izard SV Gel and PCR Clean-Up System The prepared DNA fragment was designated as Insert A. Next, a random region (a region corresponding to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing) A SPINK2 oligonucleotide containing the following was synthesized: 5'-GC AAA TAT CGT ACC CCG AAT TGT UUU UU U UUU UUU UUU VVV UUU TGT VVV UUU UUU WW W XXX CCG GTT GGT AGC GAT ATG-3' UUU, VVV, WWW and XXX are any bases selected from A, T, G and C Represents. UUU contains 18 amino acids (Ala, Glu, Gln, As) excluding Cys and Pro. p, Asn, His, Trp, Arg, Lys, Val, Leu, Ile, Phe, Ty It contains codons encoding ribonucleotides (r, Ser, Met, Gly, Thr). VVV contains 19 amino acids (Ala, Glu, Gln, Asp, As) excluding Cys. n, His, Trp, Arg, Lys, Val, Leu, Ile, Phe, Tyr, Se It contains codons encoding ribonucleotides (r, Met, Gly, Thr, Pro). WWW contains codons encoding Tyr, Ser, Phe, Leu, and Thr. There are. XXX encodes Asn, Asp, Leu, Lys, Gln, Ala, and Glu. Contains codons. Using Insert A and SPINK2 oligonucleotide as templates, -9 and 10, and PfuUltra II Fusion HS DNA Po PCR using lysine 100 (Agilent) (95°C for 20 seconds, 55°C for 2 10 cycles of incubation at 72°C for 30 seconds and 72°C for 30 seconds. Primer 9: 5'-GTTTGGTCTGTTTAGCAAATATCGTACCCC GAATTGT-3' Primer 10: 5'-GCACGGACCATTGCGAATA-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. D with the izard SV Gel and PCR Clean-Up System The prepared DNA fragment was designated Insertion B. B was used as a template, and the following primers 11 and 12 and PfuUltra II PCR using Fusion HS DNA Polymerase (95°C 20 55°C for 20 seconds, 72°C for 30 seconds) × 10 cycles were performed. Primer 11: 5'-AAAGAATTCTGATCCGCAGTTTGGTCTGT TTAGCAAATAATCGT-3' Primer 12: 5'-AAAGGCGCGCCGCACGGACCATTGCGAAT AATTTTAAT-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. D with the izard SV Gel and PCR Clean-Up System The prepared DNA fragment was designated Insert C. Insert C was controlled by The phagemid vector pPR was digested with the restriction enzymes EcoRI (NEB) and AscI (NEB). 3_stuffer_TEV was cloned into EcoRI (TAKARA) and MluI (TAKAR A) at 37°C for 5 hours or more, and then insert C into the Purified using the SV Gel and PCR Clean-Up System After subjecting the phagemid vector to agarose gel electrophoresis, the desired DNA fragment was obtained. Excise the A fragment and use Wizard SV Gel and PCR Clean-Up. The purified fragment was purified using the T4 DNA Ligand. The ligation reaction was carried out overnight at 16°C using NEB. The next day, the sample was heat-treated at 65°C for 10 minutes and then placed in an Amicon-Ultra (30k:M Desalting of the DNA was performed by filtration using a microcentrifuge.

[0167] (1-5) E. coli XL1-Bl carrying randomly mutated SPINK2 phagemid vector Preparation of ue Next, competent cells for transformation were prepared. The day before, they were incubated overnight at 37°C in 2-YT XL1-Blue (Stratagen) cultured in medium (Invitrogen) e) was inoculated into 2-YT medium and cultured at 37°C for several hours. After cooling on ice, the mixture was centrifuged (3,000 The pellet was recovered by centrifugation (g, 10 minutes, 4°C), suspended in sterilized water, and then centrifuged. The pellet was suspended in 10% glycerol, centrifuged, and then resuspended in 10% glycerol. The pellet was then suspended in 10% glycerol and centrifuged. , and were used as competent cells. Using the DNA prepared in (1-4) and competent cells, electroporation ( Transformation was carried out using a voltage of 1.97 kV and 186 μF. The mixture was incubated at 37°C for 1 hour with shaking, and then 0.1 mg / ml ampicillin (Wako Pure Chemical Industries) was added. and plated on 2-YT plates containing 2% glucose (Nacalai Tesque). The medium was then incubated at 30°C for 1 hour. The next day, the medium was incubated with 1% glucose and 15% glycerol. Colonies were collected using 2-YT solution containing 100% PEG-400 (Wako Pure Chemical Industries, Ltd.) and stored at -80°C. After electroporation, a portion of the culture medium was collected and serially diluted before plating. The next day, the number of colonies was counted to estimate the library size. , the size of the constructed library was 1.2 × 10 10 It was confirmed that it was to an extent.

[0168] (1-6) Construction of randomly mutated SPINK2 phage library The E. coli colonies constructed in (1-5) were incubated with 0.1 mg / ml ampicillin and 1% Inoculate the bacteria into 2-YT medium containing glucose and measure the OD 600nm Prepare a suspension of E. coli at 0.3 μL By culturing with shaking at 37°C, the OD 600nm Culture until the pH reaches 0.5. Add the helperphage VCSM13 (Stratagene) and mix for 37 minutes. The mixture was left to stand at 37°C for 30 minutes, and then cultured with shaking at 37°C for 30 minutes. The mixture was left to stand for 30 minutes, and then centrifuged (3,000 g, 20 minutes, 4°C) to collect the pellet. 0.1mg / ml ampicillin, 30μg / ml kanamycin (nacalai tes que), suspended in 2-YT medium containing 0.25 mM IPTG (Wako Pure Chemical Industries), and The mixture was incubated overnight at 10°C with shaking. The next day, the culture supernatant was collected by centrifugation (9,000 g, 20 min, 4°C) and diluted with 20% P olyethylene glycol 6000(nacalai tesque) Add 1 / 4 of the volume of 2.5M NaCl (Wako Pure Chemical Industries) solution and let stand at 4°C for 30 minutes. The phage particles were precipitated by PEG precipitation (hereinafter referred to as "PEG precipitation"). The phage was then centrifuged twice (9,000 g, 30 min, 4°C) and the precipitated phage was washed with PBS. A randomly mutated SPINK2 phage library was prepared by suspending the phage in PBS. The prepared phage solution was infected into E. coli XL1-Blue, and 0.1 mg / ml amplicons were added. The colonies were then plated onto 2-YT plates containing silane and 1% glucose, and the number of colonies formed was counted. The titer of the phage library was 1.8 × 10 13 phage / ml .

[0169] [Example 2] Selection of SPINK2 mutants that bind to target molecules (2-1) Liquid-phase panning method EZ-Link NHS-Chromogenic Biotin Reagent( Using a Thermo Scientific™ ELISA kit, the target protein described in (2-4) was biotinylated according to the attached instructions. It has become a The biotinylated target protein was mixed with SPINK2 mutant-displaying phages and reacted for 1 to 12 hours. After the reaction, Dynabeads M-280 Streptavidin (In Vitrogen (hereafter simply referred to as "beads") to bind biotinylated target proteins. The protein was bound to the beads and then diluted with PBS containing 0.05% Tween (hereafter referred to as "PBS-T"). After washing with AcTEV for the specified number of times, TM Protease (Invitrogen n) for 30 minutes to detect SPI bound to the biotinylated target protein on the bead surface. The phage displaying the NK2 mutant was collected. The collected phage was then transferred to E. coli XL1-Blue. Infect and plate 2-YT containing 0.1 mg / ml ampicillin and 1% glucose. The cells were seeded on a plate and cultured overnight at 30°C. The first round of liquid-phase panning involved random mutations. The SPINK2 phage library was then used in the second and subsequent rounds to identify the large phage fragments obtained in the previous round. Phages prepared from colonies of Enterococcus coli were used.

[0170] (2-2) Solid-phase panning method Nunc Maxisorp flat-bottom 96 well plate A certain amount of target protein was added to the plate (Nunc), and the plate was incubated overnight at 4°C. Immobilization was performed using Pierce NHS-Activated Agarose. Add a certain amount of target protein to Dry Resin (Thermo) and follow the instructions provided. Therefore, immobilization on agarose was carried out. The target protein-immobilized plate or agarose is used to visualize the SPINK2 mutant displaying phagocytosis. After the reaction, beads were added to the mixture, and the biotinylated target protein was detected. Protein was bound to the beads, washed a predetermined number of times with PBS-T, and then incubated with AcTEV. TM Pro By reacting with tease (Invitrogen) for 30 minutes, the biotin on the beads surface was The SPINK2 mutant-displaying phages that bound to the lysed target protein were collected. The cells were infected with E. coli XL1-Blue and treated with 0.1 mg / ml ampicillin and 1% glutamic acid. The cells were seeded on glucose-containing 2-YT plates and cultured overnight at 30°C. In the first round, a randomly mutated SPINK2 phage library was used, and in the second and subsequent rounds, In the next round, phages prepared from the E. coli colonies obtained in the previous round were used.

[0171] (2-3) Preparation of phage The E. coli colonies obtained after panning were cultured in 0.1 mg / ml ampicillin and 1% Inoculate the bacteria into 2-YT medium containing glucose and measure the OD 600nm Prepare a suspension of E. coli at 0.3 μL By culturing with shaking at 37°C, the OD 600nm Culture until the pH reaches 0.5. Add the helperphage VCSM13 (Stratagene) and mix for 37 minutes. The mixture was left to stand at 37°C for 30 minutes, and then cultured with shaking at 37°C for 30 minutes. The mixture was left to stand for 30 minutes, and then centrifuged (3,000 g, 20 minutes, 4°C) to collect the pellet. 0.1mg / ml ampicillin, 30μg / ml kanamycin (nacalai tes que), suspended in 2-YT medium containing 0.25 mM IPTG (Wako Pure Chemical Industries), and The culture was performed overnight at ℃ with shaking. The next day, the culture supernatant was collected by centrifugation and PEG precipitated. The phage solution to be used in the next round was prepared by repeating the above-mentioned centrifugation twice.

[0172] (2-4) Screening of SPINK2 mutants that bind to target molecules Using one of the following four types of target protein, perform liquid or solid phase panning for 3 to 5 rounds. The campaign was carried out. Chymotrypsin (Worthington) ·Recombinant Human Plasma Kallikrein(R&D systems) ·Recombinant Human EGFR / Fc(R&D systems: (hereinafter referred to as "EGFR / Fc") ·Recombinant Human ErbB2 / Fc(R&D systems: (Hereinafter referred to as "HER2 / Fc")

[0173] (2-5) Evaluation of SPINK2 mutant (polyclonal) binding to target proteins According to the method described in (2-3), SPINK2 mutant display cells were isolated from the E. coli group after panning. Phages were prepared. Nunc Maxisorp flat-bottom 96 well plate (Nunc) contains the target protein described in (2-4) and a negative control. IgG-Free, Protease-Free Bovine Serum Alu bmin(Jackson ImmunoResearch Laboratories : hereinafter referred to as "BSA") was added and left to stand overnight at 4°C. Next, the SPINK prepared from the coliform bacteria after panning was coated with The phage displaying the two mutants was added as a sample and allowed to stand at room temperature for 1 hour. After removing the membrane and washing with PBS-T, HRP / Anti-M13 Monoclonal Antibody was added. Conjugate (GE Healthcare) was added and left to stand at room temperature for 1 hour. Then, remove the HRP / Anti-M13 Monoclonal Conjugate. After washing with PBS-T, the POD substrate ABTS kit (Nacalai) was added. The color was developed using HRP / An and the absorbance was measured at a wavelength of 405 nm. PBS-T was used to dilute the ti-M13 Monoclonal Conjugate. Ta. The results are shown in Figures 1 to 4. Random mutation SPI was performed by panning against each target protein. SPINK2 variants (polyclones) showing target-specific binding from an NK2 library The enrichment of was confirmed by ELISA.

[0174] [Example 3] Evaluation of α-chymotrypsin binding peptides (single clones) (3-1) Construction of pET 32a (modified) Using pIRES Puro3 (Clontech) as a template, the following primers 13 and PCR using 14 and 15, and KOD-plus- (TOYOBO) (94°C 1 5 seconds, 60°C for 30 seconds, 68°C for 30 seconds) × 30 cycles). 5'-AAAGGATCCGCGAATTCATGACCGAGTACAAGCCCAC -3' 5'-AAACTCGAGTTATGCGGCCGCTCAGGCACCGGGCTTG CGG-3' The amplified fragments were subjected to agarose gel electrophoresis, and the desired DNA fragment was excised and purified. izard SV Gel and PCR Clean-Up System (Pro DNA was prepared using the ELISA kit (mega). The prepared DNA fragment and pET 32a(+)( Novagen) were used with the restriction enzymes BamHI (TAKARA) and XhoI (TA KARA) at 37°C for 1 hour or more, and after agarose gel electrophoresis, the desired DNA fragments were excised and purified using Wizard SV Gel and PCR Clean-U. The purified fragment was purified using the T4 DNA PCR System (Promega). Ligation was carried out using ligase at 16°C overnight. The transformed E. coli JM109 was transformed with the ribosomal DNA. After culturing the transformed E. coli, "pET 32a (modified)" was constructed by performing rep and sequence analysis. The procedure was carried out according to the method described in (1-2).

[0175] (3-2) α-chymotrypsin binding peptide from Escherichia coli Origami B (D Expression and purification in E3 Using the QIAGEN Plasmid Midi Kit (Qiagen), Following the manufacturer's instructions, the phagemid vector was recovered from the E. coli colonies after panning. The recovered vector and pET 32a (modified) were purified by restriction enzyme EcoRI (TAKARA ) and NotI (TAKARA) at 37°C for 1 hour or more, and After gel electrophoresis, the desired DNA fragment was excised and placed on Wizard SV Gel and Purification was performed using the PCR Clean-Up System (Promega). The fragments were ligated with T4 DNA ligase (NEB) at 16°C overnight. The ligation reaction was carried out and Escherichia coli JM109 was transformed. The procedure was carried out according to the method described in (1-2). , and transformed Escherichia coli Origami B (DE3) (Novagen), and 0.1 mg / The cells were plated onto 2-YT plates containing 1 ml ampicillin. Next, the colonies were inoculated onto 2-YT medium containing 0.1 mg / ml ampicillin and incubated at 37°C. After overnight culture, a portion of the culture was inoculated onto 2-YT medium containing 0.1 mg / ml ampicillin. And OD 600nm After that, IPTG (final concentration 1 mM) was added. The cells were then cultured overnight at 16°C to induce expression. The next day, the cells were centrifuged (3,000g , 20 minutes, 4℃) and then collected with BugBuster Master Mix (Nova The mixture was stirred at room temperature for 20 minutes and then centrifuged (10,000 g, 20 minutes, 4°C). The supernatant was collected by TALON Metal Affinity Resin (TA KARA) and stir at 4℃ for at least 1 hour to bind His tag to the resin. The target fusion protein was adsorbed. After washing the resin several times with phosphate, 300 mM NaCl, pH 7.4, Eluent (50mM Sodium Phosphate, 300mM NaCl, 150mM The His-tagged protein was recovered by adding 100 mM imidazol (pH 7.4). Furthermore, a thrombin cleavage capture kit (No The Thioredoxin tag is cleaved using a gene called vagen and added to TALON. The thioredoxin tag was removed by this procedure. Finally, the thioredoxin tag was removed by Amicon-Ultra ( The target protein was concentrated and replaced with PBS using PBS (3k). The protein (SPINK2 mutant) was subjected to SDS-PAGE under reducing and non-reducing conditions. We attempted to analyze the molecular state. The results are shown in Figures 5 and 6. All clones showed a single band under reducing and non-reducing conditions. From the results shown, it was found that the molecular state of the expressed and purified target protein was mainly composed of monomers. was confirmed.

[0176] (3-3) Target binding of α-chymotrypsin-binding peptides expressed and purified in E. coli Affinity assessment SPINK2 mutant (α-chymotrypsin bind To confirm the target binding specificity of Nunc er, evaluation was performed using ELISA. Target proteins were added to a Maxisorp flat-bottom 96 well plate. White matter chymotrypton was added and left to stand overnight at 4°C. Next, the SPINK2 mutant prepared in (3-2) was The sample was added and allowed to stand at room temperature for 1 hour. After that, the sample was removed and the mixture was washed with PBS-T. After washing, S-Tag Antibody Affinity Purified H RP conjugated (BETHYL) was added and the mixture was left to stand at room temperature for 1 hour. After removing the detection antibody solution and washing with PBS-T, add the POD substrate ABTS kit. The color was developed using HCl, and the absorbance was measured at a wavelength of 405 nm. Antibody Affinity Purified HRP conjugate d was diluted with PBS-T. The results are shown in Figures 7 and 8. All of the SPINK2 mutants prepared in E. coli were able to bind to the target. Furthermore, the ELISA showed a sigmoid curve, indicating that E The lowest and highest signal intensities detected by LISA were compared to those showing 50% of the maximum response. The binding activity of peptide No. 2 (clone 2) was calculated as EC50. The EC50 of peptide number 6 (clone 6) was 17.8 nM. It was confirmed that...

[0177] (3-4) Target specificity of α-chymotrypsin binding peptides expressed and purified in E. coli Sexual evaluation Nunc Maxisorp flat-bottom 96 well plate The target protein chymotrypsin and the negative control trypsin (PIERCE) was added and left to stand overnight at 4℃. Next, the SPINK2 mutant prepared in (3-2) was used as a sample. The mixture was then added to the plate and allowed to stand at room temperature for 1 hour. After that, the sample was removed and the plate was washed with PBS-T. S-Tag Antibody Affinity Purified HRP con The detection antibody solution was then removed and PBS was added. After washing with -T, the color was developed using the POD substrate ABTS kit and measured at a wavelength of 40 The absorbance at 5 nm was measured. PBS-T was used to dilute the purified HRP conjugated. Ta. The results are shown in Figures 9 and 10. Peptides Nos. 2 and 6 (clone 2 and 6) Although it showed no reactivity to trypsin, it reacted with the target protein chymotactic acid. It showed a strong reaction with rypsin, indicating that it has target-specific binding. Ta.

[0178] (3-5) α-chymotrypsin binding peptide expressed in E. coli and purified Otrypsin inhibitory activity evaluation Regarding the target protein chymotrypsin, the SPINK2 mutant prepared in (3-2) Variant and Pierce Quantitative Protease Assay The inhibitory activity was quantified using a kit according to the attached instructions. The results are shown in Figure 11. Both peptides Nos. 2 and 6 (clone 2 and 6) inhibited The compounds showed activity, with IC50 values ​​of 815 nM and 374 nM, respectively.

[0179] (3-6) Sequence analysis of α-chymotrypsin-binding peptides The SPINK2 mutant ( The sequence of each SPINK2 variant (α-chymotrypsin binding peptide) was analyzed. The transformed E. coli Origami B (DE3) was incubated with 0.1 mg / ml amplicons. The cells were cultured overnight at 37°C in 2-YT medium containing cillin, and the next day, the cells were transferred to QIAprep 96 Plasmid DNA was recovered from the cultures using the Turbo Miniprep Kit. Using the plasmid DNA as a template, primer 15 having the following base sequence: 5'-GTTCTGGTTCTGGCCATATGCACCATC-3' The base sequence was analyzed using the following. The analysis results of the randomized region (random region) are shown in Figure 12. All SPINK2 mutations The randomized regions of the body had different base sequences. [Industrial Applicability]

[0180] The peptide library of the present invention can be used to search for peptides that bind to various target molecules. This is useful for research into test drugs, diagnostic drugs, etc. The binding peptides are useful as test agents, diagnostic agents, etc. [Sequence List Free Text]

[0181] Sequence Listing SEQ ID NO: 1 - Random region of peptide with diversity (Figure 13) Sequence Listing SEQ ID NO: 2 - Random region of chymotrypsin-binding peptide (Figure 12: Peptide Code number 1) Sequence Listing SEQ ID NO: 3 - Random region of chymotrypsin-binding peptide (Figure 12: Peptide Code number 2) Sequence Listing SEQ ID NO: 4 - Random region of chymotrypsin binding peptide (Figure 12: Peptide Chid number 6) Sequence Listing SEQ ID NO: 5 - Random region of chymotrypsin binding peptide (Figure 12: Peptide Chid No. 7) Sequence Listing SEQ ID NO: 6 - Random region of chymotrypsin binding peptide (Figure 12: Peptide Code number 12) Sequence Listing SEQ ID NO: 7 - Random region of chymotrypsin binding peptide (Figure 12: Peptide Code number 13) Sequence Listing SEQ ID NO: 8 - Random region of chymotrypsin binding peptide (Figure 12: Peptide Code number 14) Sequence Listing SEQ ID NO: 9 - Random region of chymotrypsin binding peptide (Figure 12: Peptide Code number 17) Sequence Listing SEQ ID NO: 10 - Fragment 1 (Figure 14) Sequence number 11 in the sequence listing - fragment 2 (underlined in Figure 15) Sequence Listing SEQ ID NO: 12 - Fragment 3 (Figure 16) Sequence Listing SEQ ID NO: 13 - Fragment 5 (Figure 17) SEQ ID NO: 14 in the sequence listing - a base sequence encoding the amino acid sequence of SPINK2 (Figure 18 ) The amino acid sequence encoded by the base sequence shown in SEQ ID NO: 15 - SEQ ID NO: 14 in the sequence listing Acid sequence Sequence Listing SEQ ID NO: 16 - Base sequence of PCR template DNA including fragments 1 to 5

Claims

1. A physical population of cells containing oligonucleotides or polynucleotides comprising nucleotide sequences encoding amino acid sequences of peptides comprising amino acid sequences of peptides contained in the peptide library described below (i), wherein the peptides are not identical: (i) An amino acid sequence encoded by a base sequence in which the base sequence consisting of the 43rd base thymine to the 93rd base thymine in SEQ ID NO: 14 of the Sequence Listing is replaced with a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, wherein X １ ~X １２ is a naturally occurring amino acid other than cysteine, and in which one to five amino acids other than amino acid numbers 2 to 8 and 10 to 14 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing may be substituted, deleted, added, and / or inserted.

2. 2. The physical collection of cells of claim 1, wherein said oligonucleotide or polynucleotide is directly linked to said peptide comprising the amino acid sequence it encodes.

3. 3. The physical collection of cells of claim 1 or 2, wherein the cells are prokaryotic or eukaryotic cells.

4. The physical collection of cells of claim 3 , wherein the prokaryotic cells are Escherichia coli or Bacillus subtilis.

5. 4. The physical collection of cells of claim 3, wherein the eukaryotic cells are animal cells, insect cells, yeast, or fungi.

6. A physical population of microorganisms comprising oligonucleotides or polynucleotides comprising nucleotide sequences encoding amino acid sequences of peptides comprising amino acid sequences of peptides contained in the peptide library described in (i) below, wherein the peptides are not identical: (i) An amino acid sequence encoded by a base sequence in which the base sequence consisting of the 43rd base thymine to the 93rd base thymine in SEQ ID NO: 14 of the Sequence Listing is replaced with a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, wherein X １ ~X １２ is a naturally occurring amino acid other than cysteine, and in which one to five amino acids other than amino acid numbers 2 to 8 and 10 to 14 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing may be substituted, deleted, added, and / or inserted.

7. 7. A physical collection of microorganisms according to claim 6, characterized in that said oligonucleotide or polynucleotide is directly linked to said peptide comprising the amino acid sequence it encodes.

8. 8. A physical collection of microorganisms according to claim 6 or 7, wherein the microorganisms are viruses, phages, phage-like molecules, or particles of any of these.

9. A physical assembly of vectors comprising oligonucleotides or polynucleotides comprising nucleotide sequences encoding amino acid sequences of peptides comprising the amino acid sequences of peptides contained in the peptide library described below in (i), wherein the peptides are not identical: (i) An amino acid sequence encoded by a base sequence in which the base sequence consisting of the 43rd base thymine to the 93rd base thymine in SEQ ID NO: 14 of the Sequence Listing is replaced with a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, wherein X １ ~X １２ is a naturally occurring amino acid other than cysteine, and in which one to five amino acids other than amino acid numbers 2 to 8 and 10 to 14 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing may be substituted, deleted, added, and / or inserted.

10. 10. The physical assembly of a vector according to claim 9, wherein said oligonucleotide or polynucleotide is directly linked to said peptide comprising the amino acid sequence it encodes.

11. 11. The physical assembly of vectors of claim 9 or 10, wherein the vectors are naturally occurring or artificially created vectors.

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