Screening method, screening kit, oligopeptide, and composition

Abstract

To provide a technique for screening regardless of whether target protein has a domain capable of non-specifically binding to a peptide in a method for screening a ligand candidate peptide capable of specifically interacting with target protein from a peptide library generated based on a sequence of existing protein.SOLUTION: As a result of intensive research, inventors have found that by screening a ligand candidate peptide from a peptide library generated based on a sequence of existing protein using an antibody against a target substance of target protein that specifically interacts with the target substance, the ligand candidate peptide can be suitably screened even when the target protein has a domain capable of non-specifically binding to a peptide other than the ligand candidate peptide.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This technology relates to a screening method, a screening kit, an oligopeptide, and a composition. More specifically, it relates to a technology for suitably screening peptides (candidate ligand peptides) that can specifically interact with a target protein from a peptide library generated based on the sequence of an existing protein, and the screened peptides, etc. [Background technology]

[0002] Conventionally, techniques for screening candidate peptide ligands from peptide libraries generated based on the sequences of existing proteins have been known.

[0003] For example, Non-Patent Document 1 below discloses a method for screening peptides capable of interacting with DPP4, using a target protein, DPP4, from a peptide library generated based on the sequence of α-lactalbumin. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Peptide Array on Cellulose Support-A Screening Tool to Identify Peptides With Dipeptidyl-Peptidase IV Inhibitory Activity within the Sequence of α-Lactalbumin / Int. J. Mol. Sci. 2014,15,20846-20858; doi:10.3390 / ijms151120846 Summary of the Invention [Problem to be solved by the invention]

[0005] The main purpose of this technology is to provide a screening method for candidate ligand peptides that can specifically interact with a target protein from a peptide library generated based on the sequence of an existing protein, regardless of whether the target protein has a domain that can non-specifically bind to the peptide. [Means for solving the problem]

[0006] As a result of intensive research, the inventors have found that by using an antibody against a target substance of a target protein that specifically interacts with the target substance to screen for candidate ligand peptides from a peptide library generated based on the sequence of an existing protein, it is possible to suitably screen for candidate ligand peptides even if the target protein has a domain that can non-specifically bind to peptides other than the candidate ligand peptide.

[0007] That is, in the present technology, a ligand candidate peptide that is different from the target substance with which the target protein specifically interacts and that can specifically interact with the target protein is selected from the following: A screening method is provided in which a peptide library generated based on the sequence of an existing protein is screened using a screening antibody against the target substance. In the screening method of the present technology, the target protein may be a protein having a domain capable of non-specifically binding to a peptide, such as Dipeptidyl-Peptidase IV. After performing the screening method of the present technology, a next-stage overlapping peptide library may be created based on the sequence information of the existing protein and on the sequences of the candidate ligand peptides screened by the screening method, and the next-stage candidate ligand peptides may be screened one or more times from the next-stage overlapping peptide library using the screening antibody. In the screening method of the present technology, the candidate ligand peptide may be an oligopeptide. In the screening method of the present technology, the peptide library may be an overlapping peptide library generated based on the sequence of the existing protein. In the screening method of the present technology, the antibody for screening may be a monoclonal antibody. The present technology provides a screening kit for screening candidate ligand peptides, other than a target substance with which a target protein may specifically interact, which kit comprises a screening antibody against the target substance with which the target protein specifically interacts.

[0008] Next, the present technology provides oligopeptides identified by screening using the screening method of the present technology. The oligopeptide of the present technology may be an oligopeptide that interacts with dipeptidyl-peptidase IV, in which one or more amino acids among 2 to 4 residues from the N-terminus are cysteine, and in this case, the cysteine ​​may be reduced cysteine. The oligopeptide of the present technology may be an oligopeptide having at its N-terminus any of the sequences YPSYGLNY, KALCSEKL, LACQCLVR, LAHKALCS, MCVKKILD, ECAQKKII, LQSWMHQP, PAKSCQAQ, ACQCLVRT, LLQSWMHQ, and VPAKSCQA. The present technology further provides compositions comprising the oligopeptides of the present technology. [Brief explanation of the drawings]

[0009] [Figure 1] This technology was used to screen candidate ligand peptides that can specifically interact with Dipeptidyl-Peptidase IV from a peptide library generated based on the sequence of a milk protein. [Figure 2]This is the result of screening, using this technology, for candidate ligand peptides that can specifically interact with Dipeptidyl-Peptidase IV from the next-stage overlapping peptide library based on the sequences of candidate ligand peptides obtained through screening using this technology. [Figure 3] 1 shows the results of measuring the effect of oxidation treatment of oligopeptides on their inhibitory activity against DPP IV. DETAILED DESCRIPTION OF THE INVENTION

[0010] Preferred embodiments of the present technology will be described below. However, the embodiments shown below are examples of typical embodiments of the present technology, and the present technology is not limited to only the preferred embodiments below and can be freely modified within the scope of the present technology.

[0011] [Screening method] In the screening method of the present technology, candidate ligand peptides are selected from a peptide library generated based on the sequence of an existing protein, and a screening antibody against the target substance is used to effectively screen candidate ligand peptides, even if the target protein has a domain that can non-specifically bind to peptides other than the candidate ligand peptide.

[0012] In the present technology, a "candidate ligand peptide" refers to a peptide that is different from the target substance with which a target protein specifically interacts and with which the target protein can specifically interact. Here, a "ligand" refers to a molecule or ion with which a specific protein (the "target protein") can specifically interact, and is a substance that, through interaction with the target protein, is involved in signal transduction or metabolic pathways in the body and has physiological functions such as promoting biochemical reactions, and in the present technology refers to the "target substance." Furthermore, a "target protein" is a protein that interacts with the ligand (target substance). For example, if the target protein is an enzyme protein, a peptide or the like that can serve as a substrate for the enzyme protein would be the target substance (ligand).

[0013] In other words, a "candidate ligand peptide" is a substance that is different from a ligand, which is a target substance with which a target protein specifically interacts, but is a peptide that can specifically interact with the target protein. Therefore, although a "candidate ligand peptide" interacts with the target protein, unlike a ligand, it is not involved in signal transduction or metabolic pathways in the body, and may serve as an "inhibitor" that inhibits the biochemical reactions of the signal transduction or metabolic pathways between the target substance and the target protein.

[0014] When screening candidate peptide ligands from a peptide library using a target protein, peptides other than candidate ligand peptides may be screened due to factors such as the target protein having a domain that can nonspecifically bind to peptides. In such cases, it is highly likely that the screened peptides will not function as inhibitors of the target protein.

[0015] In contrast, in the present technology, candidate ligand peptides are screened from a peptide library using a screening antibody against the target substance instead of the target protein.

[0016] The term "antibody for screening against a target substance" refers to an antibody generated using the target substance as an antigen. Because the antibody does not have a domain that can nonspecifically bind to peptides, it can avoid screening peptides other than candidate ligand peptides from a peptide library and can suitably screen candidate ligand peptides.

[0017] The screening antibody used in the screening method of the present technology is not particularly limited. In the screening method of the present technology, for example, a monoclonal antibody or a polyclonal antibody can be used. It is preferable to use a monoclonal antibody from the viewpoint of reducing the frequency of screening for unintended peptides due to nonspecific binding and more accurately screening for peptides that interact more specifically with the target protein. Furthermore, the accuracy of screening can be further improved by using a monoclonal antibody that specifically recognizes a site involved in specific interaction with the target protein, such as the binding activity center of a ligand (target substance).

[0018] As used herein, peptides contained in a "peptide library generated based on the sequence of an existing protein" are synthesized or manufactured based on the amino acid sequence of any protein existing in nature. That is, the amino acid sequences of peptides contained in the peptide library are not based on the amino acid sequence of any protein existing in nature and are not randomly determined.

[0019] Peptides contained in a peptide library generated based on the sequence of an existing protein may be modified by known techniques, such as by amino acid substitution, deletion, or addition. Furthermore, solubility or antigen-antibody reactivity suitable for various applications may be imparted to the peptides. The peptides can be produced by known peptide synthesis methods, such as a fully automated peptide synthesizer or a method using genetic recombination in yeast, Escherichia coli, mammalian cells, or the like.

[0020] In the peptide library used in the screening method of the present technology, the peptides are preferably immobilized on a suitable solid-phase carrier. As used herein, "immobilization" refers to the concept of linking, adsorbing, encapsulating, embedding, or supporting a peptide on a solid-phase carrier. The material and shape of the solid-phase carrier are not particularly limited, and any known solid-phase carrier can be used, as long as it is insoluble in the solvent in the antigen-antibody reaction system. The shape of the solid-phase carrier can be selected appropriately depending on the intended use, and examples include test plate, bead, sphere, disk, tube, filter, etc. Flat plate shapes such as test plate, disk, and filter are preferred. Furthermore, the material can be, for example, a material typically used as a carrier for immunoassays. Specific examples include synthetic resins such as polypropylene, polystyrene, and polyacrylamide, or resins to which reactive functional groups such as sulfonic acid groups and amino groups have been introduced by known methods, glass, polysaccharides, silica gel, porous ceramics, and metal oxides. More specifically, the solid support is, for example, a cellulose membrane, a glass plate, a microwell plate, or the like.

[0021] The method for immobilizing peptides on a solid support is not particularly limited, and known methods such as physical adsorption, covalent bonding, ionic bonding, and cross-linking can be used.

[0022] In the present technology, the solid support on which the peptides are immobilized is preferably in the form of a peptide array. In this specification, the term "peptide array" refers to a solid support on which multiple peptides are immobilized.

[0023] If necessary, the solid support on which the peptide is immobilized may be treated to prevent nonspecific adsorption onto the structure. This treatment is preferably performed by coating with a blocking agent that does not impair the activity of the supported peptide. The blocking agent is not particularly limited, and examples include collagen, gelatin, skim milk, casein, and serum proteins such as BSA. Other compounds that do not react with proteins and contain both hydrophobic and hydrophilic moieties can also be used.

[0024] The peptide library used in the screening method of the present technology may be, for example, an "overlapping peptide library" in which peptides having a certain number of amino acid residues are designed based on the amino acid sequence of any protein existing in nature, with the number of amino acid residues being shifted by a specific amount, or a "random peptide library" generated by randomly degrading any protein existing in nature.

[0025] The naturally occurring proteins that form the basis of the above-mentioned arbitrary peptide library are not particularly limited, and any protein can be used depending on the business objectives. For example, when a peptide library is generated based on the sequences of proteins contained in animal or plant foodstuffs, the peptides contained in the peptide library are sequences derived from foodstuffs, and therefore can be expected to be highly safe for living organisms. Therefore, the ligand candidate peptides screened using this technology can also be expected to be highly safe.

[0026] Examples of animal-based food materials include dairy products, meat, eggs, etc., and examples of plant-based food materials include grains, vegetables, fruits, beans, seeds, etc. By screening using a peptide library generated based on the sequences of proteins contained in these food materials, highly safe candidate ligand peptides can be extracted.

[0027] In other words, the screening method of this technology efficiently screens candidate peptide ligands for a target protein from a peptide library generated based on the sequence of an existing protein, thereby efficiently identifying peptides derived from the sequence of the existing protein that could serve as inhibitors that block signal transduction and biochemical reactions in metabolic pathways mediated by the target protein. Furthermore, by using a peptide library based on proteins contained in foodstuffs, it is expected that highly safe inhibitor candidates can also be efficiently identified.

[0028] The target protein that can be the target of the screening method of the present technology is not particularly limited as long as it is a protein that can interact with a specific ligand in vivo. For example, any protein can be set as the target protein depending on the biochemical reaction of the signal transduction or metabolic pathway that you want to inhibit. As mentioned above, the screening method of the present technology can be used to effectively screen candidate ligand peptides even if the target protein is a protein that has a domain that can nonspecifically bind to a peptide.

[0029] Examples of proteins having a domain capable of non-specifically binding to a peptide include, but are not limited to, dipeptidyl-peptidase IV, angiotensin converting enzyme, ghrelin O-acyltransferase, cathepsin K, GLP-1 receptor, adrenergic receptor, dopamine receptor, adenosine receptor, serotonin receptor, prostaglandin receptor, histamine receptor, muscarinic receptor, serotonin receptor, GABA receptor, nicotinic receptor, glycine receptor, transient receptor potential channel, insulin, steroid hormone, thyroid hormone, and the like.

[0030] Candidate ligand peptides that can be screened using the screening method of the present technology are not particularly limited, but oligopeptides are often expected to be the target from the standpoints of ease of creating a peptide library, ease of interaction with the antibody used for screening, and ease of handling after screening (peptide structural stability, digestion resistance, absorbability in the body, etc.).

[0031] The upper limit of the number of amino acid residues in the oligopeptide is not particularly limited, but can be adjusted to, for example, 10 or less, 20 or less, 30 or less, etc., depending on the intended use and application. The lower limit of the number of amino acid residues is not particularly limited, but can be adjusted to, for example, 2 or more, 5 or more, 10 or more, etc.

[0032] In the screening method of the present technology, the screening can be performed using the above-mentioned screening antibody to screen for candidate ligand peptides from the peptide library based on the binding strength between the antibody and the peptides in the peptide library. The screening may be performed using only the screening antibody, or may be performed using a secondary antibody against the screening antibody.

[0033] In the screening method of the present technology, after performing the screening method of the present technology, a next-stage overlapping peptide library may be created based on the sequence information of existing proteins and using the sequences of the candidate ligand peptides screened by the screening method as a reference, and further screening of next-stage candidate ligand peptides may be performed from the next-stage overlapping peptide library using a screening antibody.

[0034] For example, an overlapping peptide library designed by shifting a specific number of amino acid residues ("shift range of residues during primary screening") based on the sequence of an existing protein is used as the peptide library used in the initial screening. Using this technology, candidate ligand peptides are screened ("primary screening") to identify regions with amino acid sequences that can specifically interact with the target protein.

[0035] Next, using the amino acid sequences of the candidate ligand peptides obtained by the first screening as a reference, a next-stage overlapping peptide library is designed, shifted by a number of amino acid residues ("second-stage residue shift") that is shorter than the shift in residues in the first screening. Further screening using this next-stage overlapping peptide library can efficiently identify candidate ligand peptides having amino acid sequences that can interact more specifically with the target protein.

[0036] Although the case where two rounds of screening are performed after the primary screening has been described, the number of rounds of screening performed after the primary screening is not particularly limited. For example, by appropriately performing an appropriate number of screening rounds by designing the range of residue shifts in the primary screening and the range of residue shifts in the secondary screening and thereafter, it is possible to efficiently identify candidate ligand peptides having amino acid sequences that can interact more specifically with the target protein.

[0037] [Screening kit] This technology can also be implemented as a screening kit for screening candidate peptide ligands, other than a target substance, with which a target protein may specifically interact, comprising a screening antibody for the target substance with which the target protein specifically interacts.

[0038] In this case, the screening kit of the present technology can be used to suitably screen for candidate ligand peptides that are different from the target substance, for example, from any peptide library designed from proteins existing in nature.Furthermore, by using a peptide library based on proteins contained in foodstuffs, it is expected that highly safe inhibitor candidates can be efficiently identified.

[0039] [Oligopeptide] As mentioned above, oligopeptides identified by screening using the screening method of the present technology can be candidates for inhibitors that inhibit biochemical reactions in signal transduction or metabolic pathways in which the target protein is involved in vivo.

[0040] For example, when the target protein is dipeptidyl-peptidase IV ("DPP IV"), an example of an oligopeptide identified by screening using the screening method of the present technology is an oligopeptide that interacts with DPP IV, in which one or more of the amino acids from the N-terminus are cysteine. By interacting with DPP IV, the oligopeptide can inhibit the degradation reaction of GLP-1 (glucagon-like peptide 1) by DPP IV.

[0041] When an oligopeptide identified by screening using the screening method of the present technology is an oligopeptide that interacts with DPP IV and in which one or more of the amino acids 2 to 4 from the N-terminus are cysteines, the cysteines are preferably reduced cysteines. The reduced cysteines can be obtained by known techniques. Specifically, for example, an oligopeptide containing cysteine ​​in its sequence can be reduced to obtain the oligopeptide containing the reduced cysteine. Furthermore, for example, when peptides are synthesized by chemical methods such as solid-phase synthesis, cysteine ​​is synthesized as reduced cysteine, and therefore, peptides containing reduced cysteine ​​synthesized by such methods may be used without oxidation.

[0042] Examples of oligopeptides that interact with DPP IV and in which one or more amino acids among the 2 to 4 residues from the N-terminus are cysteine ​​include, but are not limited to, oligopeptides having an N-terminal sequence such as MCVKKILD (SEQ ID NO: 1), ECAQKKII (SEQ ID NO: 2), and ACQCLVRT (SEQ ID NO: 3). As described above, in the oligopeptides exemplified above, by replacing one or more amino acids among the 2 to 4 residues from the N-terminus with reduced cysteine, the inhibitory activity against DPP IV can be improved.

[0043] In addition to the above, examples of oligopeptides of the present technology that have been identified by screening using the screening method of the present technology include, but are not limited to, oligopeptides having an N-terminus sequence such as YPSYGLNY (SEQ ID NO: 4), KALCSEKL (SEQ ID NO: 5), LACQCLVR (SEQ ID NO: 6), LAHKALCS (SEQ ID NO: 7), LQSWMHQP (SEQ ID NO: 8), PAKSCQAQ (SEQ ID NO: 9), LLQSWMHQ (SEQ ID NO: 10), and VPAKSCQA (SEQ ID NO: 11).

[0044] Here, the upper limit of the number of amino acid residues of an oligopeptide having the above-mentioned specific sequence at its N-terminus is not particularly limited, as long as the N-terminus contains the above-mentioned sequence. For example, the number of amino acid residues can be suitably adjusted within the ranges listed above as the number of amino acid residues of the oligopeptide of the present technology.

[0045] The oligopeptides of the present technology may be modified by known methods, such as amino acid substitution, deletion, or addition, depending on the purpose and application, as in the synthesis of reduced cysteine ​​exemplified above. It is also possible to impart solubility and antigen-antibody reactivity suitable for various applications. The oligopeptides of the present technology can be produced by known peptide synthesis methods, such as methods using fully automated peptide synthesizers, or genetic recombination in yeast, Escherichia coli, mammalian cells, etc. Alternatively, they may be produced by hydrolyzing a protein having the sequence of an oligopeptide identified by the present technology using an enzyme or the like.

[0046] The oligopeptide of the present technology may, if necessary, be in the form of a salt, preferably a physiologically acceptable acid addition salt, such as a salt of an inorganic acid (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or an organic acid (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).

[0047] The oligopeptides of the present technology can also be used as compositions that can be used as solutes in solvents, depending on the purpose and application.

[0048] The present technology can have the following configurations. [1] A ligand candidate peptide that is different from the target substance with which the target protein specifically interacts and that can specifically interact with the target protein is selected. Using a screening antibody against the target substance, A screening method that screens from a peptide library generated based on the sequence of an existing protein. [2] The screening method according to [1], wherein the target protein is a protein having a domain capable of non-specifically binding to a peptide. [3] The screening method according to [1] or [2], wherein the target protein is Dipeptidyl-Peptidase IV. [4] After performing the screening method described in any one of [1] to [3], creating a next-stage overlapping peptide library based on the sequences of the candidate ligand peptides screened by the screening method, based on the sequence information of the existing protein; From the next-stage overlapping peptide library, A screening method in which the screening antibody is used to screen candidate peptides for the next stage ligand one or more times. [5] The screening method according to any one of [1] to [4], wherein the candidate ligand peptide is an oligopeptide. [6] The screening method according to any one of [1] to [5], wherein the peptide library is an overlapping peptide library generated based on the sequence of the existing protein. [7] The screening method according to any one of [1] to [6], wherein the antibody for screening is a monoclonal antibody. [8] An oligopeptide identified by screening using the screening method according to any one of [1] to [7]. [9] Providing a screening antibody against a target substance with which the target protein specifically interacts; A screening kit for screening candidate peptides that are ligands other than the target substance and that can specifically interact with the target protein.

[10] An oligopeptide that interacts with dipeptidyl-peptidase IV, in which one or more of the amino acids in the 2 to 4 residues from the N-terminus are cysteine.

[11] The oligopeptide according to

[10] , wherein the cysteine ​​is a reduced cysteine.

[12] An oligopeptide having at its N-terminus any one of the following sequences: YPSYGLNY, KALCSEKL, LACQCLVR, LAHKALCS, MCVKKILD, ECAQKKII, LQSWMHQP, PAKSCQAQ, ACQCLVRT, LLQSWMHQ, or VPAKSCQA.

[13] A composition comprising the oligopeptide according to any one of

[10] to

[12] . [Example]

[0049] The present technology will be described in detail below based on specific examples, but the present technology is not limited to the contents of the examples shown below.

[0050] <Primary peptide library> Existing proteins include α-derived proteins from cow's milk. S1 -Casein (199 amino acid residues), α S2 The milk proteins used were -casein (207 amino acid residues), β-casein (209 amino acid residues), κ-casein (169 amino acid residues), α-lactalbumin (123 amino acid residues), and β-lactoglobulin (162 amino acid residues). Based on the amino acid sequences of these milk proteins, oligopeptides consisting of eight amino acid residues were synthesized by solid-phase synthesis. These oligopeptides were synthesized starting from the N-terminus of the milk proteins, shifting by four residues at a time, to create a primary peptide library, an overlapping peptide library.

[0051] <Antibodies for screening> The target protein was human-derived DPP IV (Dipeptidyl-Peptidase IV), and the target substance was human-derived GLP-1 (Glucagon-Like Peptide 1). The screening antibody used was a monoclonal antibody (Bioporto Diagnostics, Cat. No. ABS 033-10B) against HAEGTFTSDVS (SEQ ID NO: 12), an oligopeptide consisting of 11 amino acid residues on the N-terminus of GLP1, which is involved in the specific interaction with DPP IV.

[0052] <First screening> The screening antibodies were used to screen candidate ligand peptides from the primary peptide library. Figure 1 shows the binding scores between the peptides in the primary peptide library and the screening antibodies.

[0053] Based on the binding scores between the oligopeptides in the first peptide library and the screening antibodies, 11 peptides were selected as candidate ligand peptides. The average binding score (1st binding assay score) between these oligopeptides and the screening antibodies was 14,436.

[0054] The IC values ​​of the 11 selected oligopeptides against DPP IV were 50 The half-maximal inhibitory concentration (IC) was measured. 50 The following three oligopeptides were identified that exhibited inhibitory activity against DPP IV with a concentration of 1000 μM or less (positive hit rate: 3 / 11=27.3%). The results are shown in Table 1.

[0055] Oligopeptides that exhibit inhibitory activity against DPP IV identified by the primary screening ECAQKKII (SEQ ID NO: 2) LAHKALCS (SEQ ID NO: 7) LACQCLVR (SEQ ID NO: 6)

[0056] Among the oligopeptides that exhibit inhibitory activity against DPP IV identified by the above-mentioned first screening, ECAQKKII (SEQ ID NO: 2) and LACQCLVR (SEQ ID NO: 6) have cysteines in at least one of the two to four residues from the N-terminus, and the cysteines they contain are reduced cysteines.

[0057] [Table 1]

[0058] <Secondary peptide library> Next, starting from the sequences of the 11 oligopeptides selected in the first screening, oligopeptides were synthesized by solid-phase synthesis, shifting one residue forward for up to two residues and one residue backward for up to three residues, to create a second peptide library, the next-stage overlapping peptide library (number of oligopeptides: 11 x 5 = 55). The number of amino acid residues in the oligopeptides contained in the second peptide library was eight, the same as in the first peptide library.

[0059] <Second screening> The screening antibodies were used to screen candidate ligand peptides from the secondary peptide library. Figure 2 shows the binding scores between the oligopeptides in the secondary peptide library and the screening antibodies.

[0060] Based on the binding scores between the peptides in the second peptide library and the screening antibodies, 15 oligopeptides were selected as candidate ligand peptides. The average binding score between these oligopeptides and the screening antibodies (second binding assay score) was 21,831.

[0061] The IC values ​​of the 15 selected oligopeptides against DPP IV were 50 As a result, IC 50 The following eight peptides were identified that exhibited inhibitory activity against DPP IV with a concentration of 1000 μM or less (positive hit rate: 8 / 15=53.3%). The results are shown in Table 2.

[0062] Oligopeptides showing inhibitory activity against DPP IV identified by the secondary screening MCVKKILD (SEQ ID NO: 1) LQSWMHQP (SEQ ID NO: 8) KALCSEKL (SEQ ID NO: 5) YPSYGLNY (SEQ ID NO: 4) PAKSCQAQ (SEQ ID NO: 9) ACQCLVRT (SEQ ID NO: 3) LLQSWMHQ (SEQ ID NO: 10) VPAKSCQA (SEQ ID NO: 11)

[0063] Among the oligopeptides exhibiting inhibitory activity against DPP IV identified by the above-mentioned secondary screening, MCVKKILD (SEQ ID NO: 1), KALCSEKL (SEQ ID NO: 5), and ACQCLVRT (SEQ ID NO: 3), in which one or more amino acids among 2 to 4 residues from the N-terminus are cysteine, had reduced cysteines.

[0064] [Table 2]

[0065] Next, a total of nine oligopeptide solutions, namely ECAQKKII (sequence number 2), LACQCLVR (sequence number 6), MCVKKILD (sequence number 1), KALCSEKL (sequence number 5), and ACQCLVRT (sequence number 3), in which one or more amino acids among the two to four residues from the N-terminus are cysteine, and HAEGTFTSDVS (sequence number 12), LQSWMHQP (sequence number 8), YPSYGLNY (sequence number 4), and LLQSWMHQ (sequence number 10), which do not have cysteine ​​residues, were treated under the following conditions.

[0066] Each of the above oligopeptides was adjusted to a final concentration of 10 mg / mL in the reaction solution, and 10% v / v dimethyl sulfoxide (DMSO) was added to the solution, followed by treatment at 37°C for 24 hours.

[0067] Figure 3 shows the activity inhibition rates of the degradation reaction for the nine oligopeptides mentioned above, both before and after treatment. The intensity of fluorescence (decomposition intensity) emitted by the fluorescent substrate (a substrate that emits fluorescence upon decomposition) by DPP IV without the addition of these oligopeptides is set to 100% (the activity inhibition rate of the degradation reaction is 0%). The activity inhibition rate in this case indicates the rate of change in the decomposition intensity of the fluorescent substrate by DPP IV due to the addition of the oligopeptide. In other words, 0% indicates that the added oligopeptide did not inhibit the degradation reaction at all. The test was performed in triplicate.

[0068] The decomposition intensity in the above degradation reaction was measured as the fluorescence value of the fluorescent substrate when a fluorescent substrate was added to a DPP IV solution with a final concentration of 1.5 mU / mL to give a final concentration of 8.3 μM and incubated for 15 minutes at 37° C. Each oligopeptide was added at a concentration that resulted in a 50% DPP IV inhibition rate compared to the DMSO-untreated peptide.

[0069] 3, it can be seen that for five oligopeptides in which one or more amino acids among the two to four residues from the N-terminus are cysteine, the activity inhibition rate of the treated oligopeptide (oxidized oligopeptide) is lower than that of the untreated oligopeptide (reduced oligopeptide). In other words, it can be seen that when an oligopeptide contains a cysteine ​​among the two to four residues from the N-terminus, converting the cysteine ​​to a reduced cysteine ​​can improve the inhibitory activity against DPP IV.

[0070] In the graph of Figure 3, the p-values ​​for the data of ECAQKKII (sequence number 2), LACQCLVR (sequence number 6), MCVKKILD (sequence number 1) and KALCSEKL (sequence number 5) are p<0.05, and the p-value for the data of ACQCLVRT (sequence number 3) is p<0.01, so it can be said that the activity inhibitory effect shown in Figure 3 is a statistically significant value.

[0071] [Sequence table] <110> MORINAGA MILK INDUSTRY CO., LTD. <120> Screening methods, screening kits, oligopeptides and compositions <130> P-002560 <160> 12

[0072] <210> SEQ ID NO:1 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 1 MCVKKILD

[0073] <210> SEQ ID NO:2 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 2 ECAQKKII

[0074] <210> SEQ ID NO:3 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 3 ACQCLVRT

[0075] <210> SEQ ID NO:4 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 4 YPSYGLNY

[0076] <210> SEQ ID NO:5 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 5 KALCSEKL

[0077] <210> SEQ ID NO:6 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 6 LACQCLVR

[0078] <210> SEQ ID NO:7 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 7 LAHKALCS

[0079] <210> SEQ ID NO:8 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 8 LQSWMHQP

[0080] <210> SEQ ID NO:9 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 9 PAKSCQAQ

[0081] <210> SEQ ID NO:10 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 10 LLQSWMHQ

[0082] <210> SEQ ID NO:11 <211> Length 8 <212> Type PRT <213> Organism Bos taurus <400> Sequence 11 VPAKSCQA

[0083] <210> SEQ ID NO:12 <211> Length 11 <212> Type PRT <213> Organism Homo sapiens <400> Sequence 12 HAEGTFTSDVS

Claims

1. A ligand candidate peptide that is different from the target substance with which the target protein specifically interacts and that can specifically interact with the target protein is selected. Using a screening antibody against the target substance, A screening method that screens from a peptide library generated based on the sequence of an existing protein.

2. The screening method according to claim 1, wherein the target protein is a protein having a domain capable of non-specifically binding to a peptide.

3. The screening method according to claim 1, wherein the target protein is Dipeptidyl-Peptidase IV.

4. After carrying out the screening method according to claim 1, creating a next-stage overlapping peptide library based on the sequences of the candidate ligand peptides screened by the screening method, based on the sequence information of the existing protein; From the next-stage overlapping peptide library, A screening method in which screening of candidate peptides for the next stage ligand is carried out one or more times using the screening antibody.

5. The screening method according to claim 1 , wherein the candidate ligand peptide is an oligopeptide.

6. The screening method according to claim 1, wherein the peptide library is an overlapping peptide library generated based on the sequence of the existing protein.

7. The screening method according to claim 1, wherein the antibody for screening is a monoclonal antibody.

8. An oligopeptide identified by screening using the screening method according to any one of claims 1 to 7.

9. A screening antibody for a target substance with which the target protein specifically interacts is provided. A screening kit for screening peptides other than the target substance, which are candidate ligand peptides with which the target protein can specifically interact.

10. An oligopeptide that interacts with Dipeptidyl-Peptidase IV, in which one or more amino acids among 2 to 4 residues from the N-terminus are cysteine.

11. The oligopeptide of claim 10, wherein the cysteine ​​is a reduced cysteine.

12. An oligopeptide having at its N-terminus any one of the following sequences: YPSYGLNY, KALCSEKL, LACQCLVR, LAHKALCS, MCVKKILD, ECAQKKII, LQSWMHQP, PAKSCQAQ, ACQCLVRT, LLQSWMHQ, and VPAKSCQA.

13. A composition comprising the oligopeptide of any one of claims 10 to 12.

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