Peptides, kits, and methods for producing peptides

Abstract

To provide a novel peptide having beta-2-microglobulin binding activity.SOLUTION: A peptide having binding activity to beta-2-microglobulin according to one aspect of the present invention comprises an amino acid sequence including the following sequence (1): YWX1X2 (1), in the sequence (1), X1 is an acidic amino acid and X2 is a basic amino acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to peptides, kits, and methods for producing peptides. [Background technology]

[0002] Beta-2-microglobulin (β2-M) is a low molecular weight protein consisting of 99 amino acids with a molecular weight of 11,800, and is expressed on the surface of most somatic cells except red blood cells. The concentration of β2-M in serum increases with a decrease in glomerular filtration rate, and is therefore useful as an indicator of renal glomerular damage. β2-M is also a uremic substance that causes dialysis amyloidosis. Therefore, there is a demand for the development of technology to detect or quantify β2-M.

[0003] As an example of such a technique, Patent Document 1 describes a monoclonal antibody that has the activity of binding to β2-M. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2022-121306 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, the monoclonal antibody described in Patent Document 1 has a molecular weight of about tens of thousands to hundreds of thousands, making it difficult to produce it by chemical synthesis. Therefore, the method for producing the monoclonal antibody mainly uses biosynthesis, which tends to be expensive. In addition, monoclonal antibodies having a molecular weight of about tens of thousands to hundreds of thousands have poor thermal stability, and therefore require refrigerated storage.

[0006] One aspect of the present invention has been made in consideration of the above-mentioned problems, and an object of the present invention is to provide a novel peptide having the activity of binding to beta-2-microglobulin. [Means for solving the problem]

[0007] In order to solve the above problems, a peptide having an activity of binding to beta-2-microglobulin according to one embodiment of the present invention comprises an amino acid sequence including the following sequence (1): YWX1X2(1) In the above sequence (1), X1 is an acidic amino acid and X2 is a basic amino acid.

[0008] Furthermore, a kit for producing a complex between a peptide according to an aspect of the present invention and beta-2-microglobulin comprises a peptide according to an aspect of the present invention.

[0009] Furthermore, a method for producing a peptide having binding activity to beta-2-microglobulin according to one embodiment of the present invention includes a nucleic acid library preparation step of preparing a library of nucleic acids, a display library preparation step of preparing a library of displays which are complexes of the nucleic acids and peptides encoded by the nucleic acids, a contact step of contacting the display library with beta-2-microglobulin, a separation step of separating the display from the complex of the display and beta-2-microglobulin, and an amplification step of amplifying the nucleic acid contained in the separated display. Effect of the Invention

[0010] According to one aspect of the present invention, the object is to provide a novel peptide having an activity of binding to beta-2-microglobulin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 〔peptide〕 Hereinafter, one embodiment of the present invention will be described in detail. In this specification, a peptide is a compound in which two or more amino acids are bonded together by amide bonds. The number of amino acid residues contained in a peptide is two or more, and the upper limit is not particularly limited. The number of amino acid residues may be, for example, 80 or less. In this specification, an amino acid sequence refers to a sequence consisting of two or more amino acid residues. In this specification, an amino acid sequence is described with the left side being the N-terminus and the right side being the C-terminus.

[0012] As used herein, the term "amino acid residue" refers to a residue derived from an amino acid that constitutes a peptide. The amino acid includes natural amino acids, including the 20 types of amino acids (i.e., common amino acids) that constitute proteins in living organisms, and non-natural amino acids. Each amino acid residue is independently in the L-form or D-form.

[0013] Each amino acid residue may be independently chemically modified.The chemical modification is not particularly limited as long as it is a modification compatible with living organisms, and includes, for example, phosphorylation, amidation and PEGylation of the carboxyl group present in the amino acid residue, and N-acetylation, N-formylation, N-acylation, PEGylation, biotinylation and fluorescent labeling of the amino group present in the amino acid residue.

[0014] A peptide having an activity of binding to beta-2-microglobulin according to one embodiment of the present invention comprises an amino acid sequence including the following sequence (1): YWX1X2(1) In sequence (1), X1 is an acidic amino acid and X2 is a basic amino acid. Hereinafter, when simply referred to as "amino acid sequence" in this specification, it refers to an amino acid sequence including sequence (1).

[0015] In this specification, beta-2-microglobulin (β2-M) refers to a single-chain polypeptide that comprises a human leukocyte antigen (HLA) as its light chain. The activity of binding to β2-M (hereinafter, sometimes referred to as "β2-M binding activity") can be evaluated by methods known to those skilled in the art. For example, the β2-M binding activity can be evaluated using the dissociation constant K D However, the evaluation can be based on the above, and may be performed using ELISA, immunochromatography, or biolayer interference.

[0016] In sequence (1), X1 is an acidic amino acid. Acidic amino acids include aspartic acid (D) and glutamic acid (E), as well as any natural and unnatural amino acids having an isoelectric point (pI) of 5.7 or less, preferably 3.5 or less. In order to further improve the β2-M binding activity of the peptide, X1 is preferably D or E, and more preferably D.

[0017] In sequence (1), X2 is a basic amino acid. The basic amino acid includes lysine (K), arginine (R), and histidine (H), as well as any natural amino acid or unnatural amino acid having an isoelectric point of 7.5 or more. In order to further improve the β2-M binding activity of the peptide, X2 is preferably R or H, and more preferably R.

[0018] To further improve the β2-M binding activity of the peptide, the amino acid sequence preferably contains the following sequence (2) comprising X3 at the C-terminus of sequence (1), where sequence (1) is included in the amino acid sequence as a part of sequence (2).

[0019] YWX1X2X3(2)

[0020] In sequence (2), X3 is an aromatic ring-containing amino acid. Examples of aromatic ring-containing amino acids include histidine (H), phenylalanine (F), tyrosine (Y), and tryptophan (W), as well as any natural and non-natural amino acids that contain an aromatic ring in the molecule. In order to further improve the β2-M binding activity of the peptide, X3 is preferably H or F, and more preferably H. The amino acids X1 and X2 contained in sequence (2) are as described above for sequence (1).

[0021] To further improve the β2-M binding activity of the peptide, the amino acid sequence is preferably represented by the following sequence (3) (SEQ ID NO:4) which contains sequence (1), where the amino acid sequence consists of sequence (3) and sequence (1) is included as a part of sequence (3).

[0022] CHFYWX1X2HX5KKX4FHX5C (3)

[0023] In sequence (3), X4 is tyrosine (Y), histidine (H), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q). In order to further improve the β2-M binding activity of the peptide, X4 is preferably tyrosine, asparagine, aspartic acid or histidine, more preferably tyrosine. X5 is each independently any amino acid, preferably any natural amino acid, more preferably any essential amino acid. The amino acids X1 and X2 contained in sequence (3) are as described above for sequence (1).

[0024] To further improve the β2-M binding activity of the peptide, the amino acid sequence is preferably represented by SEQ ID NO: 1, 2 or 3, more preferably SEQ ID NO: 1 or 3. Of these, the amino acid sequence represented by SEQ ID NO: 1 is a preferred specific example of the amino acid sequence represented by the above-mentioned sequence (3). The amino acid sequence represented by SEQ ID NO: 1, 2 or 3 contains sequence (1) as a part thereof.

[0025] CHFYWDRHVKKYFHLC (SEQ ID NO: 1) CYFYWDRHVKKYFHLC (SEQ ID NO:2) CLYWDRFTWPRVLKVC (SEQ ID NO:3)

[0026] Alternatively, to further improve the β2-M binding activity of the peptide, the amino acid sequence may have a specific sequence identity to the sequence shown in SEQ ID NO: 1, 2 or 3. The sequence identity is preferably 80% or 81% or more, more preferably 85% or 87% or more, even more preferably 90% or 93% or more, and even more preferably 95% or more. The higher the sequence identity, the more likely the β2-M binding activity of the peptide is to be improved.

[0027] In the present specification, the sequence identity of amino acid sequences can be determined using the BLASTX program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing amino acid sequences using BLASTX, the parameters are, for example, score=50 and wordlength=3.

[0028] The amino acid sequence may be linear or may include a cyclic structure. The amino acid sequence preferably further includes at least one amino acid residue on each of the N-terminus and C-terminus of the sequence (1), and includes a cyclic structure formed by a bond between at least one of the amino acid residues further included on the N-terminus and at least one of the amino acid residues further included on the C-terminus. Such a cyclic structure tends to stabilize the three-dimensional structure of the peptide and further improve the β2-M binding activity of the peptide. The cyclic structure is formed by the whole or a part of the amino acid sequence. When the cyclic structure is formed by the whole amino acid sequence, typically, the N-terminus amino acid residue and the C-terminus amino acid residue of the amino acid sequence are bonded to each other.

[0029] The bond forming the cyclic structure may be a direct bond between amino acid residues such as a disulfide bond or a diselenide bond, or a bond via a crosslinking agent such as bismaleimide ethane or α,α'-dibromoxylene. From the viewpoint of bond stability and ease of bond formation, the cyclic structure is preferably formed by a disulfide bond between amino acid residues containing an S atom. The disulfide bond may be realized by the amino acid sequence further containing at least one amino acid residue containing an S atom at each of the N-terminus and C-terminus of sequence (1). Examples of amino acid residues containing an S atom include, but are not limited to, cysteine, and may be amino acid residues other than cysteine ​​substituted to have -S. The cyclic structure formed by a disulfide bond between amino acid residues contained in the amino acid sequence further improves the β2-M binding activity of the peptide.

[0030] The number of amino acid residues contained in the amino acid sequence, including the number of amino acid residues contained in sequence (1), is preferably 10 or more, more preferably 12 or more, and even more preferably 14 or more. The more amino acid residues there are, the more the β2-M binding activity and binding selectivity tend to improve. The peptide according to one embodiment of the present invention exhibits sufficient β2-M binding activity due to the short amino acid sequence represented by sequence (1). Therefore, it should be noted that the above-mentioned preferred number of amino acid residues does not limit the present invention. The number of amino acid residues contained in the amino acid sequence, including the number of amino acid residues contained in sequence (1), is preferably 50 or less, more preferably 40 or less, even more preferably 30 or less, and even more preferably 20 or less. The fewer the number of amino acid residues, the more the thermal stability and ease of production of the peptide can be improved.

[0031] The peptide may consist of an amino acid sequence containing sequence (1), or may further contain other moieties in addition to the amino acid sequence. The other moieties include, but are not limited to, an amino acid sequence other than the amino acid sequence containing sequence (1); a sugar chain; and a linker structure such as PEG and a carbon chain. Here, the amino acid sequence other than the amino acid sequence containing sequence (1) may be adjacent to the amino acid sequence containing sequence (1) or may be separated. The number of amino acid residues contained in the amino acid sequence other than the amino acid sequence containing sequence (1) may be, for example, 1 or more, 2 or more, or 4 or more, and may be 30 or less, 25 or less, or 20 or less, but is not limited thereto. The other moieties are appropriately selected within a range that does not impair the β2-M binding activity of the peptide. The other members may be present at either the N-terminal side or the C-terminal side of the amino acid sequence containing sequence (1), or may be present at both.

[0032] In the peptide, since the amino acid sequence including the sequence (1) exhibits β2-M binding activity, other components can be appropriately selected depending on the application of the peptide. Therefore, the proportion (based on molecular weight) of the amino acid sequence including the sequence (1) in the peptide is not particularly limited.

[0033] The molecular weight of the peptide is preferably 400 or more. The larger the molecular weight of the peptide, the more improved the β2-M binding activity and binding selectivity. The molecular weight of the peptide is preferably 8000 or less, more preferably 3000 or less. The molecular weight of the peptide is typically much smaller than that of the conventional technology, which not only enables peptide production at a lower cost, but also improves the thermal stability of the peptide, allowing storage at a higher temperature than before, such as room temperature.

[0034] Kit for producing a conjugate of a peptide with beta-2-microglobulin Other aspects of the present invention will be described below, in which, for the sake of convenience, the description of members having the same functions as those described above will not be repeated.

[0035] A kit for producing a complex between a peptide according to an embodiment of the present invention and beta-2-microglobulin includes the peptide according to an embodiment of the present invention. By using the kit according to an embodiment of the present invention, the peptide according to an embodiment of the present invention can be bound to β2-M to produce a complex. For example, the produced complex may be used to detect or quantify β2-M.

[0036] The kit may further include any of the following optional components: reagents used to generate a complex between a peptide and β2-M, such as a β2-M standard solution, a dilution buffer, and a washing solution; reagents for detecting or quantifying the generated complex, such as a detection probe; instruments used to generate or detect or quantify the complex, such as a container; and instructions for use of the kit. The instructions may be a paper medium on which instructions for the user are printed, or may be an electronic medium that stores instructions for the user. The instructions may also be a paper or electronic medium that, instead of explaining how to use the kit, explains how to access the instructions for the user (such as the URL of a medium that can be viewed on the Internet).

[0037] [Method for producing peptides] A method for producing a peptide having beta-2-microglobulin binding activity according to one embodiment of the present invention includes a nucleic acid library preparation step of preparing a library of nucleic acids, a display library preparation step of preparing a display library which is a complex between nucleic acids and peptides encoded by the nucleic acids, a contact step of contacting the display library with beta-2-microglobulin, a separation step of separating the display from the complex between the display and beta-2-microglobulin, and an amplification step of amplifying the nucleic acids contained in the separated display.

[0038] [Nucleic acid library preparation process] The nucleic acid library preparation step is a step of preparing a library of nucleic acids. The nucleic acid is single-stranded or double-stranded DNA or RNA, typically double-stranded DNA. A nucleic acid library is a collection of multiple nucleic acids, each of which has an independent random nucleic acid sequence. The nucleic acid may have a primer binding region at each of the 5'-end and 3'-end, or may have a random region (i.e., a random nucleic acid sequence) between the primer binding regions at both ends. In addition to the random region and the primer binding region, the nucleic acid may include at least one of a promoter region, an enhancer region, a His tag region, and a linker hybridization region.

[0039] The number of bases contained in the nucleic acid (i.e., the base length) can be appropriately selected and is, for example, 90 to 300. The number of bases contained in each of the primer binding regions on the 5'-end and 3'-end of the nucleic acid can also be appropriately selected and is, for example, 20 to 150.

[0040] The nucleic acid library preparation step can be carried out using methods known in the art for preparing a library of nucleic acids, each of which independently has a random region.

[0041] [Display library preparation process] The display library preparation step is a step of preparing a display library. A display is a complex of a nucleic acid (genotype) contained in the prepared nucleic acid library and a peptide (phenotype) encoded by the nucleic acid. A display library is a collection of displays, and each display contained in the collection independently contains a nucleic acid and a peptide corresponding to the nucleic acid.

[0042] The display library preparation process can be carried out by transcription of nucleic acid, formation of a complex between the nucleic acid or its transcript and a linker, and then reverse transcription and translation of the nucleic acid or its transcript contained in the complex. As a more specific example, when a double-stranded DNA library is used as the nucleic acid library, the double-stranded DNA is subjected to a transcription process, the resulting transcript mRNA is linked to an appropriate linker such as a puromycin linker, and the resulting mRNA-linker complex is subjected to reverse transcription and translation to obtain a DNA-mRNA-linker-peptide complex (i.e., display). By such operations, a display library corresponding to the nucleic acid library can be obtained.

[0043] The display library preparation step may include a cyclization step of cyclizing peptides contained in the display after the display is formed. The cyclization step can be performed by forming a bond, such as a disulfide bond or a diselenide bond, between amino acid residues in the amino acid sequence contained in the peptide. When a disulfide bond is formed, oxidized glutathione (GSSG) and disulfide bond isomerase may be added to the system.

[0044] [Contact process] The contacting step is a step of contacting the display library with beta-2-microglobulin. According to the contacting step, among the displays contained in the library, displays having an activity of binding to β2-M form a complex with β2-M. The contact between the display library and β2-M can be performed, for example, by mixing the display library and β2-M in a solution.

[0045] The contact step may include an isolation step of isolating the complex between the display and β2-M after the contact between the display library and β2-M. The isolation step may be performed using a method known in the art. For example, isolation may be performed using an isolation probe, in which case β2-M may be immobilized to the isolation probe in advance during the above-mentioned contact. The isolation probe is a probe used to isolate the complex between the display and β2-M from a mixture of the display library and β2-M, and may be, for example, a magnetic bead. The immobilization may be achieved, for example, by binding between streptavidin and biotin.

[0046] The contact step may include a removal step of contacting the display library with a removal probe and removing the resulting complex before contacting the display library with β2-M. Here, the removal probe is a probe having the same characteristics as the isolation probe described above, except that β2-M is not immobilized, and examples of such probes include magnetic beads. By removing the complex formed by the display library and the removal probe, displays obtained in the display library preparation step that have an activity of nonspecifically binding to the isolation probe without β2-M are removed in advance from the display library. Therefore, the removal step enables more efficient isolation of displays that have an activity of specifically binding to β2-M in the isolation step. In the contact step, the removal step may be performed once or more, for example, twice or three times.

[0047] [Separation process] The separation step is a step of separating the display from the complex of the display and beta-2-microglobulin. According to the separation step, the display can be separated and collected from the complex formed in the contact step to obtain a display having the activity of binding to β2-M. The separation can be performed by a method known in the art, for example, but is not limited to, the denaturation of β2-M through the addition of an alkaline solution, solvent replacement, or thermal denaturation by heating.

[0048] The method for the separation step can be appropriately selected depending on whether or not the isolation step and removal step are performed in the contact step. In other words, since the display having the activity of binding to β2-M can be separated and recovered in the separation step, the presence or absence of the isolation step and removal step in the contact step can be selected by those skilled in the art as necessary.

[0049] [Amplification step] The amplification step is a step of amplifying the nucleic acid contained in the separated display. The amplification step can be performed, for example, by further separating the nucleic acid from the display and subjecting the obtained nucleic acid to PCR treatment, but is not limited thereto. It is preferable that the conditions of the amplification step are set so that the nucleic acid sequence of the nucleic acid obtained by amplification contains a predetermined ratio of mutations compared to the nucleic acid sequence of the nucleic acid contained in the separated display.

[0050] [Repeated series of steps] In the method for producing a peptide according to one embodiment of the present invention, a series of steps from the display library preparation step to the amplification step is preferably repeated two or more times. The more the series of steps are repeated, the more the selective extraction of a display having β2-M binding activity from the display library in the contact step and the separation step, and the mutation of the nucleic acid sequence in the amplification step are repeated. Therefore, a display having higher β2-M binding activity and a peptide contained therein are produced. With one series of steps being one round, the method for producing a peptide preferably includes two or more rounds, more preferably four or more rounds.

[0051] [First measurement process] The method for producing a peptide according to one embodiment of the present invention preferably further comprises a first measurement step of measuring the binding activity of the separated display to beta-2-microglobulin after the separation step. In the separation step, the display is separated and collected in the form of a library of displays composed of nucleic acids having various random regions and peptides encoded by the nucleic acids. The first measurement step is a step of individually measuring the β2-M binding activity of each display composed of peptides containing specific amino acid sequences in the library.

[0052] In the first measurement step, nucleic acids are separated and collected from the display library, and the products are subjected to sequence analysis using a next-generation sequencer (NGS) to grasp the degree of convergence of the nucleic acid library. Among the analyzed nucleic acids, nucleic acids that account for a high proportion of the nucleic acid library and similar nucleic acids that code for the same peptide as the nucleic acid are selected, amplified, and subjected to measurement of β2-M binding activity.

[0053] The β2-M binding activity can be measured by subjecting the selected nucleic acid to the same operations as those in the display library preparation step and contact step described above, measuring the amount of display removed using the removal probe and the amount of display isolated using the isolation probe, and calculating the ratio of the two. The amount of display can be measured by nucleic acid quantification using qPCR.

[0054] The first measuring step may be repeated two or more times as part of a sequence of steps, or may be performed only once after the sequence of steps.

[0055] [Second measurement process] The method for producing a peptide according to one embodiment of the present invention preferably further comprises, after the separation step, a second measurement step of measuring the beta-2-microglobulin-binding activity of the peptide encoded by the nucleic acid contained in the separated display. The second measurement step allows evaluation of the beta-2-M binding activity of the peptide itself, which is independent of components (e.g., nucleic acid, mRNA, and linker) other than the peptide constituting the display.

[0056] The second measurement step can be carried out by producing a peptide encoded by a nucleic acid selected using sequence analysis in the same manner as in the first measurement step, by biosynthesis or chemical synthesis, and subjecting the product to a method known to those skilled in the art, such as surface plasmon resonance measurement, ELISA, immunochromatography, or biolayer interference.

[0057] The second measurement step may be repeated two or more times as part of a sequence of steps, or may be performed only once after the sequence of steps.

[0058] As can be understood from the above explanation, the method for producing a peptide according to one embodiment of the present invention may be an embodiment of in vitro selection using a cDNA display method and employing β2-M as a target antigen for screening.

[0059] [Method for producing peptide according to another embodiment] The method for producing a peptide according to the present invention is not limited to the above-mentioned embodiment. A peptide containing the above-mentioned amino acid sequence may be produced by a production method using biosynthesis or chemical synthesis. Here, biosynthesis includes in vitro synthesis using a cell-free translation system and in vivo synthesis using a host cell into which a vector containing a nucleic acid sequence encoding the peptide has been introduced. Chemical synthesis includes solid-phase peptide synthesis, and organic synthesis using 9-fluorenylmethoxycarbonyl and tertiary-butoxycarbonyl groups, etc.

[0060] 〔summary〕 The peptide having an activity of binding to beta-2-microglobulin according to the first aspect of the present invention comprises an amino acid sequence including the following sequence (1): YWX1X2(1) In the above sequence (1), X1 is an acidic amino acid and X2 is a basic amino acid.

[0061] The peptide according to aspect 2 of the present invention is the peptide according to aspect 1 above, characterized in that the amino acid sequence comprises 10 or more amino acid residues including the sequence (1).

[0062] The peptide of aspect 3 of the present invention is characterized in that, in aspect 1 or 2 described above, the amino acid sequence is represented by SEQ ID NO: 1, 2 or 3, or the amino acid sequence has 80% or more sequence identity to the sequence represented by SEQ ID NO: 1, 2 or 3.

[0063] The peptide according to aspect 4 of the present invention is characterized in that in any one of aspects 1 to 3 described above, X1 in the sequence (1) is D or E.

[0064] The peptide according to aspect 5 of the present invention is characterized in that, in any one of aspects 1 to 4 described above, in the sequence (1), X2 is R or H.

[0065] A peptide according to a sixth aspect of the present invention is any one of the first to fifth aspects described above, wherein the amino acid sequence comprises the following sequence (2) comprising X3 at the C-terminus of the sequence (1): YWX1X2X3(2) In the sequence (2), X3 is an aromatic ring-containing amino acid.

[0066] The peptide according to the seventh aspect of the present invention is characterized in that, in the above-mentioned sixth aspect, X3 in the sequence (2) is H or F.

[0067] A peptide according to an eighth aspect of the present invention is any one of the above-mentioned first to seventh aspects, wherein the amino acid sequence is represented by the following sequence (3) (SEQ ID NO: 4) containing the sequence (1), CHFYWX1X2HX5KKX4FHX5C (3) In the sequence (3), X4 is Y, H, D, E, Q or N, and each X5 is independently any amino acid.

[0068] A peptide according to a ninth aspect of the present invention is any one of the above-mentioned aspects 1 to 8, wherein the amino acid sequence further comprises at least one amino acid residue on each of the N-terminus and C-terminus of the sequence (1), and comprises a cyclic structure formed by a bond between at least one of the amino acid residues further comprised on the N-terminus and at least one of the amino acid residues further comprised on the C-terminus. It is characterized by:

[0069] The peptide of aspect 10 of the present invention is characterized in that, in the above-mentioned aspect 9, the bond between the amino acid residue on the N-terminus side and the amino acid residue on the C-terminus side is a disulfide bond between amino acid residues containing S atoms.

[0070] A kit for producing a complex of the peptide according to the eleventh aspect of the present invention and beta-2-microglobulin includes the peptide according to any one of the first to tenth aspects described above.

[0071] A method for producing a peptide having binding activity to beta-2-microglobulin, according to aspect 12 of the present invention, includes a nucleic acid library preparation step of preparing a library of nucleic acids, a display library preparation step of preparing a library of displays which are complexes of the nucleic acids and peptides encoded by the nucleic acids, a contact step of contacting the display library with beta-2-microglobulin, a separation step of separating the display from the complex of the display and beta-2-microglobulin, and an amplification step of amplifying the nucleic acid contained in the separated display.

[0072] A peptide production method according to a thirteenth aspect of the present invention is characterized in that, in the above-mentioned twelfth aspect, the display library preparation step to the amplification step are regarded as a series of steps, and the series of steps is repeated two or more times.

[0073] The method for producing a peptide according to aspect 14 of the present invention is characterized in that, in the above-mentioned aspect 12 or 13, it further comprises a first measurement step of measuring the binding activity of the separated display to beta-2-microglobulin after the separation step.

[0074] The method for producing a peptide according to aspect 15 of the present invention, in any one of aspects 12 to 14 described above, is characterized in that it further comprises, after the separation step, a second measurement step of measuring the binding activity of the peptide encoded by the nucleic acid contained in the separated display to beta-2-microglobulin.

[0075] [Additional Notes] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. EXAMPLES

[0076] [Production of peptides] As an example of the present invention, the production of a peptide will be described below.

[0077] Preparation of beta-2-microglobulin A DNA fragment encoding the amino acid sequence (sequence number 17) of beta-2-microglobulin (β2-M) with an Avi-Tag (registered trademark) sequence added to the N-terminus was synthesized and introduced into pET-16b (Merck Millipore) to prepare an expression vector.

[0078] The expression vector was used to transform E. coli BL21(DE3) strain (Merck Millipore) by heat shock method, and cultured overnight at 28°C in LB agar medium containing ampicillin at a final concentration of 100 μg / mL. The transformant was subcultured at 28°C in 200 mL of 2×YT medium containing ampicillin at a final concentration of 100 μg / mL, and protein expression was induced with isopropyl-β-D-galactopyranoside at a final concentration of 1 mM at a cell density of OD=0.8, and further cultured for 12 hours. The cells were collected by centrifugation at 7000 rpm for 20 minutes, and the obtained cells were suspended in TBS buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4). The cells were ultrasonically disrupted using Astrason3000 (misonix), and the gene expression product was collected as inclusion bodies by centrifugation at 12000 rpm for 20 minutes.

[0079] The collected β2-M inclusion bodies were suspended in a washing solution (50 mM Tris-HCl, 200 mM NaCl, 4% Triton X-100) and collected by centrifugation at 12000 rpm for 20 minutes. The above washing procedure was repeated three times. The washed β2-M inclusion bodies were solubilized with a protein denaturant (6 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.4), diluted with 200 times the amount of protein refolding solution (20 mM Tris-HCl, pH 7.4), and stirred at 4°C for 12 hours to refold the denatured protein. The solution was concentrated by ultrafiltration, dialyzed against 20 mM Tris-HCl, pH 7.0, and purified by anion exchange chromatography using Resource Q (GE Healthcare).

[0080] The purified β2-M was biotinylated using a BirA biotin-protein ligase standard reaction kit (AVIDITY) according to the attached manual. After the reaction, unreacted biotin was removed using a Zeba Micro Spin Desalting Column (ThermoFisher SCIENTIFIC).

[0081] [Nucleic acid library preparation process] A nucleic acid library was prepared using a reverse random nucleic acid library provided by Tsukuba Oligo Co., Ltd. as a template. The obtained nucleic acid library is a collection of double-stranded DNA consisting of a random region consisting of 42 bases, a primer binding region on the 5' end side of the random region, and a linker hybridization region on the 3' end side of the random region. Here, the primer binding region contains a T7 promoter and enhancer.

[0082] Reverse random nucleic acid library template: CNNK14C (T7g10M-GS3) (93mer) (SEQ ID NO: 18) 5'-GCTGCCGCTGCCGCTGCCGCAMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNGCAACCCATATGTATATCTCCTTCTTAAAG-3' N represents any one of A, T, G and C. M represents either A or C. Primer sequence: T7g10M.F46 (46mer) (SEQ ID NO: 19) 5'-TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATA-3' Primer sequence: cnvK NewYtag.R39 (39mer) (SEQ ID NO: 20) 5'-TTTCCACGCCGCCCCCCGTCCTGCTGCCGCTGCCGCTGC-3'

[0083] Extension PCR was performed using PrimeStar HS DNA Polymerase (Takara Bio) as the kit, following the protocol. The extension reaction was performed in a reaction volume of 25 μL. As primers, 2.5 μL of the above-mentioned T7g10M.F46 and CNNK14C (T7g10M-GS3) were used at a final concentration of 1.0 μM. The extension reaction program was (a) 94°C / 60 seconds, (b) 55°C / 60 seconds, and (c) 72°C / 60 seconds, with steps (b) to (c) repeated for 5 cycles.

[0084] PCR was performed in a reaction volume of 1000 μL using 25 μL of the extension reaction solution prepared as described above as a template. Primers used were T7g10M.F46 and cnvK NewYtag.R39 at a final concentration of 0.25 μM. The PCR reaction program was (a) 94°C / 60 sec, (b) 94°C / 40 sec, (c) 68°C / 40 sec, and (d) 72°C / 40 sec, with steps (b) to (d) repeated for 6 cycles.

[0085] The PCR reaction solution was purified by extraction with phenol and chloroform, followed by precipitation with ethanol, and the PCR product obtained by dissolving in milliQ (100 μL) was used in the subsequent step.

[0086] [Display library preparation process] (Transcript) RiboMAX (T7) (Promega) was used as the kit, and transcription was performed overnight or more at 37°C in a reaction volume of 1000µL, following the protocol, using 100µL of PCR product as a template. 15µL of DNase was added to the transcription solution and reacted at 37°C for 30 minutes. The transcription product mRNA was purified by electrophoresing the solution on 8M urea 8% PAGE and excising and purifying the gel piece containing the target mRNA.

[0087] (Linkage between mRNA and puromycin linker) NaCl solution (1.0 M, 8.0 μL), Tris-HCl solution (pH 7.4, 0.25 M, 8.0 μL), puromycin linker (20 μM, 16 μL) (synthesis of which was outsourced and carried out by Tsukuba Oligo Co., Ltd.), and mRNA (20 μM, 8.0 μL) were mixed to prepare 40 μL of ligation reaction solution (5.0 μL for round 2 and after). Annealing was carried out using a PCR machine under the following conditions: 90°C / 120 seconds, then 70°C / 60 seconds, then 25°C / 30 seconds, then incubation at 4°C. Next, in order to form a photocrosslink between cnvK having a puromycin linker in its main chain and mRNA, 405 mJ of UV light with a wavelength of 366 nm was irradiated using a UVP Crosslinker (analytickjena), and 40 μL of mRNA-linker complex (5.0 μL for round 2 and after) was obtained.

[0088] Puromycin linker 5'-TTTCCAKGCCGCCCCCGX(Y)CCT-3' Where: Y: Crosslinker (EMCS)-Y1-5'-TCTCTCZZCC-3'-Y2 K:cnvK X: Amino C6-dT Y1: Thiol C6 Y2: Puromycin Z:Spacer18 The amino group of X is cross-linked with the thiol group of Y1 using the cross-linking agent EMCS.

[0089] (translation) The translation was performed using PUREfrex1.0 (Gene Frontier) as the kit, following the product protocol. 150 pmol of the mRNA-linker complex (only for round 1; 6.0 pmol for round 2 and later) was mixed with various solutions provided in the kit so that the reaction volume was 150 μL (only for round 1; reaction volume 6.0 μL for round 2 and later). This mixture was incubated at 37°C for 30 minutes and at 25°C for 12 minutes. Furthermore, EDTA solution (500 mM, so that the mixture volume was 156 μL for round 1, 0.2 μL for round 2 and later) was added to the mixture, which was then incubated again at 37°C for 5 minutes to obtain the mRNA-linker peptide complex (final volume 6.2 μL for round 2 and later).

[0090] (Reverse Transcription) The kit used was ReverTraAce (Toyobo). 5x ReverTraAce Buffer (43.9 μL), dNTP (25 mM, 8.8 μL), ReverTraAce (11 μL), and 156 μL of mRNA-linker peptide complex (5.2 μL for round 2 and later) were mixed. The mixture was incubated at 42°C for 1 hour to carry out a reverse transcription reaction, synthesizing a DNA-mRNA-linker-peptide complex, i.e., a display. The resulting display is contained in the solution as a display library.

[0091] (Cyclization process) A cyclization step was performed to form disulfide bonds in the peptides contained in the display. DsbC Set (Gene Frontier) was used as the kit. In order to remove DTT contained in 5x ReverTraAce Buffer from the solution containing the display, the total volume of the solution was adjusted to 250 μL (25 μL from round 2 onwards) with selection buffer (50 mM pH 7.6 Tris-HCl, 150 mM NaCl, 0.05% Tween 20), and buffer exchange was performed using Sephadex G-25 (Sigma-Aldrich). At this time, selection buffer was used as the exchange buffer. GSSG (60 mM, 13.8 μL (1.4 μL from round 2 onwards)) and DsbC (320 μM, 13.0 μL (1.3 μL from round 2 onwards)) were mixed with the buffer-exchanged display solution. The mixture was incubated at 37° C. for 30 minutes to allow the cyclization reaction to proceed, and the peptides were cyclized. BSA (13.0 μL in round 1, 1.3 μL in round 2 and after) was added, and the total volume of the display solution was adjusted to 300 μL (45 μL in round 2 and after) with selection buffer. Quantification of the display in this mixture was performed by qPCR.

[0092] [Contact process] As part of the contacting step, a removing step and an isolating step were performed.

[0093] (Removal process) The magnetic beads used in the removal step were prepared as follows: 45.4 μL of streptavidin-modified magnetic beads (Invitrogen TM 100 μL of selection buffer was added to Dynabeads MyOne Streptavidin C1 (Product name: Dynabeads MyOne Streptavidin C1) by pipetting and suspended. The beads were left to stand on a magnetic plate for 1 minute, and the supernatant was discarded (washing procedure). This washing procedure was repeated twice in total, and the resulting magnetic bead solution was dispensed in 22.7 μL portions.

[0094] Biotin solution (500 μM, 2.3 μL) was added to one of the dispensed portions. The mixture was incubated at 25° C. for 30 minutes with gentle stirring. The mixture was left to stand on a magnetic plate for 1 minute, and the supernatant was discarded. Selection buffer was added and suspended by pipetting. This washing procedure was repeated twice in total. The mixture was then mixed with the washed magnetic bead solution (22.7 μL) without the biotin solution to prepare beads for the removal step (45.4 μL) for use in the subsequent processing.

[0095] 45 μL of the display library solution and a predetermined amount of the bead solution for the removal step were mixed and suspended by pipetting. The mixture was stirred at room temperature for 10 minutes, left to stand on a magnetic plate for 1 minute, and the supernatant was collected (removal operation). This operation was repeated twice. The display nonspecifically adsorbed to the magnetic beads was removed together with the precipitated magnetic beads.

[0096] (Contacting the display library with beta-2-microglobulin) β2-M immobilized magnetic beads were prepared as follows: 37.5 μL (Round 1 only; 5.6 μL for Round 2 and after) of streptavidin-modified magnetic beads (Invitrogen TM 100μL of selection buffer was added to the beads (Dynabeads MyOne Streptavidin C1, product name: Dynabeads MyOne, Inc.) and suspended by pipetting. The beads were left to stand on a magnetic plate for 1 minute, and the supernatant was discarded (washing operation). A designated site biotinylated β2-M solution (17.9μM, 16.7μL in round 1, 2.5μL in round 2 and after) was added, stirred at room temperature for 30 minutes, and a biotin solution (50μM, 7.3μL in round 1, 1.1μL in round 2 and after) was added and incubated at room temperature for 15 minutes to block unreacted streptavidin. After leaving the beads to stand on a magnetic plate for 1 minute, the precipitate was collected and washed three times with 100μL of selection buffer to obtain β2-M immobilized magnetic beads.

[0097] In round 1, the removal step was not performed, and the display library solution obtained by the reverse transcription step was used as is. In rounds 2 to 6, the supernatant obtained by the removal step was used as the display library solution. 300 μL (for round 1; 45 μL for rounds 2 and after) of the display library solution and a predetermined amount of V P The β2-M immobilized magnetic bead solution was mixed with β2-M immobilized magnetic beads solution and stirred at room temperature for 30 minutes to bind the display to the β2-M immobilized on the magnetic beads. The volume V of the β2-M immobilized magnetic bead solution per round was P is shown in Table 1.

[0098] [Isolation step] The mixture was left on a magnetic plate for 2 minutes, the supernatant was removed, and the precipitate was washed three times with 100 μL of selection buffer. This resulted in the isolation of the complex between display and β2-M. The recovery rate R P are shown in Table 1. P is calculated by the following formula: R P (%) = (amount of the display substance contained in the solution recovered in the isolation step in one series of steps, mol) / (amount of the display substance used in the contact step in one series of steps, mol) × 100

[0099] [Separation process] To the β2-M immobilized magnetic beads obtained in the isolation process, 400 μL of PCR reaction solution (milliQ (294 μL), 5x Prime STAR Buffer (Mg + ) (88 μL), dNTP (2.5 mM, 35.2 μL), T7g10M.F46 (10 μM, 11 μL), cnvK NewYtag.R39 (10 μM, 11 μL) (for round 1. Adjust the total volume to 100 μL from round 2 onwards)) was added as an elution buffer, and the mixture was incubated at 95 ° C for 5 minutes to separate the display from the complex, and the eluate was collected.

[0100] [Amplification step] The eluate was subjected to PCR treatment using PrimeStar HS DNA Polymerase (Takara Bio). The PCR reaction program was (a) 94°C / 300 seconds, (b) 94°C / 10 seconds, (c) 60°C / 15 seconds, and (d) 72°C / 30 seconds. Steps (b) to (d) were repeated for a cycle of Ct value calculated from quantitative PCR + 2, and the reaction was carried out at 72°C for 1 minute, followed by cooling to 10°C. The obtained PCR product was purified by extraction with phenol and chloroform, and precipitation with ethanol.

[0101] [Series of processes] The above-mentioned series of steps including the display library preparation step, contact step, separation step, and amplification step were repeated six times. The conditions and results of rounds 1 to 6 are shown in Table 1.

[0102] [Table 1]

[0103] In Table 1, R P The higher the R, the higher the β2-M binding activity of the display. P / R N The higher the R, the higher the specific β2-M binding activity of the display. As shown in Table 1, the R P / R N was more than 100, which was a higher value compared to rounds 1 to 4. This result suggests that peptides with β2-M binding activity were obtained as a convergence result of in vitro selection.

[0104] [First measurement process] (Identification of amino acid sequence) After the amplification steps of rounds 5 and 6 were completed, we requested FASMAC to carry out next-generation sequencing analysis. The preparation of the sequencing samples was carried out according to the sample preparation method for next-generation sequencing analysis by FASMAC. A region encoding a peptide was selected from the analyzed nucleic acid sequence, and the encoded amino acid sequence was identified. The results are shown in Table 2. In Table 2, the amino acid sequence of the peptide indicates the entire identified amino acid sequence. The percentage of the number of molecules indicates the percentage of the amino acid sequence relative to the total amount of amino acid sequence encoded by the analyzed nucleic acid molecule. The sequence identity indicates the sequence identity of the identified amino acid sequence to any one of SEQ ID NOs: 1 to 3, and the number in parentheses indicates the sequence number to be compared.

[0105] [Table 2]

[0106] The peptides whose amino acid sequences were identified, shown in Table 2, were peptides recovered in both the separation steps of rounds 5 and 6, and the results in Table 2 suggested that all of the peptides had β2-M binding activity. It should be noted that the relationship between the molecular number ratios of peptides in each round does not suggest the relationship between the molecular number ratios of peptides in each round. In addition, peptides whose molecular number ratios increased from round 5 to round 6 were peptides amplified through in vitro selection, and therefore were expected to have stronger β2-M binding activity. In addition, the identified amino acid sequences contained the common motif sequence YWX1X2 (sequence (1)), suggesting that convergence occurred due to in vitro selection.

[0107] (Simple binding activity evaluation) The β2-M binding activity of the displays containing each of peptides 1 to 3 shown in Table 2 was measured. The displays used were prepared using the DNA shown in SEQ ID NO: 21 for peptide 1, the DNA shown in SEQ ID NO: 22 for peptide 2, and the DNA shown in SEQ ID NO: 23 for peptide 3, in the same manner as in the display preparation step described above. Here, the following samples were prepared: (1) a display containing a peptide containing a cyclic structure formed by a disulfide bond, (2) a display containing a peptide not containing the cyclic structure, and (3) a display not containing a peptide (DNA-mRNA-linker complex). The prepared displays were subjected to the same operation as in the contact step described above. The results are shown in Table 3.

[0108] [Table 3]

[0109] As shown in Table 3, (1) display with cyclic structure and (2) display without cyclic structure showed high R P / R N These results suggest that these displays have β2-M binding activity. Among them, (1) the display with a cyclic structure showed a particularly high R P / R N The R values ​​indicated that the cyclic structure contributes to the stronger β2-M binding activity. P / R N This suggests that the β2-M binding activity of (1) the display with a cyclic structure and (2) the display without a cyclic structure is derived from the peptide, not from other components such as the linker, among the components that make up the display.

[0110] [Second measurement process] (β2-M binding activity assessed by surface plasmon resonance measurement) The measurement device used was Biacore T200 (GE Healthcare). Each of peptides 1 to 3 shown in Table 2 was immobilized on a sensor chip SA (GE Healthcare), and the dissociation constant K of the peptide with β2-M was measured by surface plasmon resonance measurement. D (=dissociation rate constant k d / binding rate constant k a ) was measured. Data processing was performed using Biacore T200 Evaluation Software Ver.2.0 (GE Healthcare). The measurement results are shown in Table 4.

[0111] [Table 4]

[0112] As shown in Table 4, peptides 1 to 3 were each 10 -7 ~10 -8 It showed a dissociation constant of order 1 and had β2-M binding activity.

[0113] The above results demonstrated that the peptide containing an amino acid sequence including sequence (1) has the activity of binding to β2-M. [Industrial Applicability]

[0114] The present invention can be utilized for the detection or quantification of beta-2-microglobulin.

Claims

1. The amino acid sequence includes the following sequence (1): YWX １ X ２ (1) In the sequence (1), X １ is an acidic amino acid, and X ２ is a basic amino acid, A peptide having the activity of binding to beta-2-microglobulin.

2. The amino acid sequence contains 10 or more amino acid residues including the sequence (1).

2. The peptide of claim 1.

3. The amino acid sequence is represented by SEQ ID NO: 1, 2 or 3, or The amino acid sequence has 80% or more sequence identity to the sequence represented by SEQ ID NO: 1, 2 or 3.

3. A peptide according to claim 1 or 2.

4. In the sequence (1), X １ is D or E; 3. A peptide according to claim 1 or 2.

5. In the sequence (1), X ２ is R or H; 3. A peptide according to claim 1 or 2.

6. The amino acid sequence has X at the C-terminus of the sequence (1). ３ The following sequence (2) is provided: YWX １ X ２ X ３ (2) In the sequence (2), X ３ is an aromatic ring-containing amino acid; 3. A peptide according to claim 1 or 2.

7. In the sequence (2), X ３ is H or F; The peptide according to claim 6.

8. The amino acid sequence is represented by the following sequence (3) (SEQ ID NO: 4), which contains the sequence (1): CHFYWX １ X ２ HX ５ KKX ４ FHX ５ C (3) In the sequence (3), X ４ is Y, H, D, E, Q or N; X ５ are each independently any amino acid; 3. A peptide according to claim 1 or 2.

9. The amino acid sequence further comprises at least one amino acid residue on each of the N-terminus and C-terminus of the sequence (1); The amino acid sequence further comprises a cyclic structure formed by a bond between at least one of the amino acid residues further included on the N-terminal side and at least one of the amino acid residues further included on the C-terminal side.

3. A peptide according to claim 1 or 2.

10. the bond between the amino acid residue on the N-terminus side and the amino acid residue on the C-terminus side is a disulfide bond between amino acid residues containing an S atom; 10. The peptide of claim 9.

11. 3. The peptide of claim 1 or 2. A kit for producing a conjugate of said peptide with beta-2-microglobulin.

12. A method for producing the peptide according to claim 1, comprising the steps of: a nucleic acid library preparation step of preparing a library of nucleic acids; a display library preparation step of preparing a display library which is a complex of the nucleic acid and a peptide encoded by the nucleic acid; contacting the library of displays with beta-2-microglobulin; separating the display from a complex of the display and beta-2-microglobulin; an amplification step for amplifying the nucleic acid contained in the separated display; Including, A method for producing a peptide having beta-2-microglobulin binding activity.

13. The display library preparation step to the amplification step are regarded as a series of steps, and the series of steps is repeated two or more times. A method for producing the peptide according to claim 12.

14. After the separation step, the method further comprises a first measurement step of measuring the activity of the separated display to bind to beta-2-microglobulin. A method for producing the peptide according to claim 12 or 13.

15. After the separation step, the method further comprises a second measurement step of measuring the beta-2-microglobulin binding activity of a peptide encoded by a nucleic acid contained in the separated display. A method for producing the peptide according to claim 14.

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