Method for producing gelled protein

Abstract

To provide methods for producing gelatinized proteins in a short time.SOLUTION: Disclosed is a method for obtaining a gelatinized product of protein (A) via step (1), where the protein (A) has a specific amino acid sequence in which 2-200 of an amino acid sequence (X) are consecutively linked where the amino acid sequence (X) is at least one of VPGVG sequence (1), GVGVP sequence (4), GPP sequence, GAP sequence and GAHGPAGPK sequence (3), where in the step (1), the temperature of a mixture of the protein (A), water and a C1-C4 alcohol is in a range of 0-80°C.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a protein gel. [Background technology]

[0002] A known method for regenerating tissue at a wound site involves gelling (changing a specific protein into a water-containing state that loses fluidity) at the affected site and using the gel as a tissue regeneration material (Patent Document 1). However, since the method described in Patent Document 1 takes a long time to gel, there has been a demand for a simple method for gelling in a short time. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2012 / 111438 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for producing a protein gel in a short period of time. [Means for solving the problem]

[0005] The present inventors have conducted extensive research to solve the above problems and have arrived at the present invention. That is, the present invention provides a method for producing a gel of protein (A) via step (1), comprising the steps of: the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and the polypeptide chains (Y') in the protein (A) is 1 to 100, the polypeptide chain (Y) is a polypeptide chain having 2 to 200 consecutive amino acids of at least one amino acid sequence (X) selected from the amino acid sequence VPGVG (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP (4) shown in SEQ ID NO: 4, the amino acid sequence GPP, the GAP, and the amino acid sequence GAHGPAGPK (3) shown in SEQ ID NO: 3; the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of the lysines and arginines is 1 to 100, the total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A) is 20 to 90% of the total number of amino acids in the protein (A); the amino acid sequence (X') is an amino acid sequence in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine, The protein (A) has a GAGAGS sequence (2), which is the amino acid sequence shown in SEQ ID NO: 2, the ratio of the number of the GAGAGS sequence (2) to the total number of the amino acid sequence (X) and the amino acid sequence (X') in one molecule of the protein (A) (GAGAGS sequence (2):total number of the amino acid sequence (X) and the amino acid sequence (X')) is 1:0.5 to 1:20; In this production method, the step (1) is a step of adjusting the temperature of a mixture containing the protein (A), water, and an alcohol having 1 to 4 carbon atoms to 0 to 80°C. [Effects of the Invention]

[0006] By using the production method of the present invention, a protein gel can be produced in a short period of time. DETAILED DESCRIPTION OF THE INVENTION

[0007] The production method of the present invention is a method for producing a gel of protein (A) through step (1), in which a gel of protein (A) is obtained, and step (1) is a step of adjusting the temperature of a mixture containing protein (A), water, and an alcohol having 1 to 4 carbon atoms to a temperature of 0 to 80°C. the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), The total number of the polypeptide chains (Y) and the polypeptide chains (Y') in the protein (A) is 1 to 100. The polypeptide chain (Y) is a polypeptide chain having 2 to 200 consecutive amino acids of at least one amino acid sequence (X) selected from the VPGVG sequence (1) shown in SEQ ID NO: 1, the GVGVP sequence (4) shown in SEQ ID NO: 4, the GPP sequence, the GAP sequence, and the GAHGPAGPK sequence (3) shown in SEQ ID NO: 3. The polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of the lysines and arginines is 1 to 100. The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A) is 20 to 90% of the total number of amino acids in the protein (A), and the amino acid sequence (X') is an amino acid sequence in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine.

[0008] In the present invention, gelation of a protein refers to a change in a state in which the protein contains water and loses fluidity. The gelation of protein (A) in the production method of the present invention is presumed to proceed according to the following mechanism. Protein (A) aggregates through hydrogen bonds, but when heated to an appropriate temperature, the hydrogen bonds are broken, and the highly hydrophilic polypeptide chains (Y) and (Y') incorporate water into their structures, forming hydrogen bonds again, which is thought to lead to gelation.

[0009] As described above, the protein (A) has the polypeptide chain (Y) and / or the polypeptide chain (Y'). Therefore, the gel of the protein (A) has excellent biocompatibility and a suitable elasticity that can accommodate the expansion and contraction of the affected area to which the gel of the present invention is applied.

[0010] The amino acid sequence (X) constituting the polypeptide chain (Y) may be of one type or of two or more types.

[0011] As the amino acid sequence (X), the VPGVG sequence (1) and the GVGVP sequence (4) are preferred from the viewpoints of biocompatibility and gelation efficiency.

[0012] Specific examples of the polypeptide chain (Y) include (VPGVG) b Array, (GVGVP) c Sequence and (GAHGPAGPK) d In addition, b to d each represent the number of consecutive amino acid sequences (X) and are integers of 2 to 200. When one molecule of protein (A) contains a plurality of polypeptide chains (Y), the polypeptide chains (Y) may be the same or different, and are represented by (VPGVG). b Array, (GVGVP) c Sequence and (GAHGPAGPK) d The sequence may have one or more types selected from the group consisting of sequences. Furthermore, when the protein (A) contains multiple polypeptide chains (Y), the number of consecutive amino acid sequences (X) may be the same or different for each polypeptide chain (Y). That is, the protein (A) may contain multiple polypeptide chains (Y) in which the numbers b to d of consecutive amino acid sequences (X) are the same, or multiple polypeptide chains (Y) in which b to d are different. From the viewpoint of biocompatibility and gelation efficiency, the polypeptide chain (Y) is (VPGVG). b Sequence and (GVGVP) c The sequence is preferred.

[0013] The polypeptide chain (Y) is a polypeptide chain having 2 to 200 consecutive amino acid sequences (X) (the above b to d are 2 to 200). From the viewpoints of water solubility, biocompatibility, and gelation efficiency, the number of consecutive amino acid sequences (X) is preferably 2 to 100 (the above b to d are 2 to 100), more preferably 2 to 50 (the above b to d are 2 to 50), and particularly preferably 2 to 40 (the above b to d are 2 to 40).

[0014] The polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of substituted lysines and arginines is 1 to 100.

[0015] Whether or not a protein (A) is a polypeptide chain (Y') is determined by whether or not the polypeptide chain (Y) results when all lysines (K) and arginines (R) in the sequence of the protein (A) are replaced with other amino acids [glycine (G), alanine (A), valine (V), proline (P), or histidine (H)].

[0016] In the polypeptide chain (Y'), the proportion of substituted lysine and / or arginine is preferably 0.06% to 5%, more preferably 0.5 to 5%, and particularly preferably 1 to 5%, from the viewpoints of water solubility, biocompatibility, and gelation efficiency.

[0017] The polypeptide chain (Y') may also contain an amino acid sequence (X') in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine. Furthermore, the amino acid sequence (X) and / or the amino acid sequence (X') constituting the polypeptide chain (Y') may each be of one type or two or more types.

[0018] Specific examples of the amino acid sequence (X') include the GKGVP sequence (7) shown in SEQ ID NO: 7, the GKGKP sequence (8) shown in SEQ ID NO: 8, the GKGRP sequence (9) shown in SEQ ID NO: 9, and the GRGRP sequence (10) shown in SEQ ID NO: 10. From the viewpoints of biocompatibility and gelation efficiency, the amino acid sequence (X') is preferably at least one sequence selected from the group consisting of the GKGVP sequence (7), the GKGKP sequence (8), and the GRGRP sequence (10), and more preferably the GKGVP sequence (7) and the GKGKP sequence (8).

[0019] The total number of polypeptide chains (Y) and polypeptide chains (Y') in one molecule of protein (A) is 1 to 100. The total number of these chains is preferably 1 to 80, and more preferably 1 to 60. The total number of polypeptide chains (Y) and polypeptide chains (Y') in one molecule of protein (A) within the above range is preferred from the viewpoints of water solubility, biocompatibility, and gelation efficiency.

[0020] When the protein (A) contains polypeptide chains (Y) with different types and / or different numbers of consecutive amino acid sequences (X), each is counted as one, and the number of polypeptide chains (Y) is the sum of the total. The same applies to polypeptide chains (Y').

[0021] In the method for producing a gel of the present invention, the total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A) is 20 to 90% of the total number of amino acids in the protein (A). If the above ratio is less than 20%, the gelling performance, which will be described in detail later, will deteriorate, and if it exceeds 90%, the water solubility will deteriorate. From the viewpoints of solubility in water, biocompatibility, and gelling efficiency, the above ratio is preferably 25 to 90%, more preferably 52.5 to 90%, and even more preferably 55 to 90%. The above ratio can be determined using a protein sequencer. Specifically, it can be determined by the following measurement method. <Method for measuring the ratio of the total number of amino acids constituting amino acid sequence (X) and amino acid sequence (X') to the total number of amino acids in protein (A)> Protein (A) is decomposed to approximately 30 residues or less using two or more cleavage methods that can cleave at specific amino acid residues. The resulting fragments are then separated by high-performance liquid chromatography (HPLC) and the amino acid sequence is read using a protein sequencer. The resulting amino acid sequence is then subjected to peptide mapping to determine the complete sequence of protein (A). The ratio of the total number of amino acids in all amino acid sequences (X) and all amino acid sequences (X') to the total number of amino acids in protein (A) is then calculated using the following formula: The percentage (%) of the total number of amino acids in all amino acid sequences (X) constituting protein (A) and all amino acid sequences (X') constituting protein (A) = [{number of amino acid sequences (X)} x {number of amino acids in amino acid sequence (X)} + {number of amino acid sequences (X')} x {number of amino acids in amino acid (X')}] / {total number of amino acids in protein (A)} x 100

[0022] Protein (A) has the GAGAGS sequence (2), which is the amino acid sequence shown in SEQ ID NO:2. When the protein (A) has a polypeptide chain (S) with a continuous GAGAGS sequence (2), from the viewpoints of biocompatibility and gelation efficiency, the polypeptide chain (S) is preferably a polypeptide chain (S1) with 2 to 200 continuous GAGAGS sequences (2). In the polypeptide chain (S1), the number of consecutive GAGAGS sequences (2) is preferably 2 to 50, more preferably 2 to 40, particularly preferably 2 to 30, and most preferably 2 to 10, from the viewpoint of biocompatibility of the gel.

[0023] In protein (A), the ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) [{number of GAGAGS sequences (2) in protein (A) × 6} / {total number of amino acids in protein (A)} × 100] is preferably 5 to 70%, more preferably 5 to 50%, and particularly preferably 5 to 45%, from the viewpoints of biocompatibility and gelation efficiency. The ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) can be determined using a protein sequencer. Specifically, it is determined by the following measurement method.

[0024] <The ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A)> Protein (A) is decomposed to approximately 30 residues or less using two or more cleavage methods that can cleave at specific amino acid residues. After separation by high-performance liquid chromatography (HPLC), the amino acid sequence is read using a protein sequencer. The obtained amino acid sequence is subjected to peptide mapping to determine the complete sequence of protein (A). The ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) is then calculated using the following formula: The ratio (%) of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) = [{number of GAGAGS sequences (2) × 6} / {total number of amino acids in protein (A)}] × 100

[0025] When the protein (A) has a total of two or more polypeptide chains selected from the group consisting of polypeptide chain (Y), polypeptide chain (Y'), GAGAGS sequence (2), and polypeptide chain (S), an intervening amino acid sequence (Z) may be present between these chains. The intervening amino acid sequence (Z) is a peptide sequence in which one or more amino acids are linked, and is not a GAGAGS sequence (2), an amino acid sequence (X), or an amino acid sequence (X'). From the viewpoints of biocompatibility and gelation efficiency, the number of amino acids constituting the intervening amino acid sequence (Z) is preferably 1 to 30, more preferably 1 to 15, and particularly preferably 1 to 10. Specific examples of the intervening amino acid sequence (Z) include the VAAGY sequence (11) shown in SEQ ID NO: 11, the GAAGY sequence (12) shown in SEQ ID NO: 12, and the LGP sequence. The ratio of the number of amino acids in all intervening amino acid sequences (Z) to the total number of amino acids in protein (A) [Σ{(number of amino acids in intervening amino acid sequences (Z)) × (number of intervening amino acid sequences (Z))} / {total number of amino acids in protein (A)} × 100] is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%, from the viewpoints of biocompatibility and gelation efficiency.

[0026] From the viewpoint of biocompatibility and gelation efficiency, the protein (A) may have a terminal amino acid sequence (T) at its terminus in addition to the GAGAGS sequence (2), the amino acid sequence (X), the amino acid sequence (X'), and the intervening amino acid sequence (Z). The terminal amino acid sequence (T) may be present at one or both termini of the protein (A). The terminal amino acid sequence (T) does not include the purification tag described below. The terminal structure of the protein (A) is preferably a structure in which a terminal amino acid sequence (T) is bound to a polypeptide chain (Y). The terminal amino acid sequence (T) is a peptide sequence in which one or more amino acids are bound, and is a peptide sequence other than the GAGAGS sequence (2), the amino acid sequence (X), and the amino acid sequence (X'). From the viewpoints of biocompatibility and gelation efficiency, the number of amino acids constituting the terminal amino acid sequence (T) is preferably 1 to 100, more preferably 1 to 50, and particularly preferably 1 to 40. Specific examples of the terminal amino acid sequence (T) include the amino acid sequence MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM (13) shown in SEQ ID NO: 13.

[0027] The ratio of the number of amino acids in the terminal amino acid sequence (T) to the total number of amino acids in the protein (A) is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%, from the viewpoints of biocompatibility and gelation efficiency.

[0028] As described below, protein (A) may be produced using bacteria by biotechnology techniques. In such cases, to facilitate the purification or detection of the expressed protein (A), the protein (A) may contain, in addition to the terminal amino acid sequence (T), a protein or peptide having a specific amino acid sequence at the N- or C-terminus (hereinafter referred to as a "purification tag"). Affinity purification tags are used as purification tags. Examples of such purification tags include the 6xHis tag consisting of polyhistidine, V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag09™, CruzTag22™, CruzTag41™, Glu-Glu tag, Ha.11 tag, and KT3 tag. Below are examples of combinations of each purification tag (i) and a ligand (ii) that recognizes and binds to that tag. (i-1) Glutathione-S-transferase (GTS) (ii-1) Glutathione (i-2) Maltose-binding protein (MBP) (ii-2) Amylose (i-3) HQ tag (ii-3) Nickel (i-4) Myc tag (ii-4) Anti-Myc antibody (i-5) HA tag (ii-5) Anti-HA antibody (i-6) FLAG tag (ii-6) Anti-FLAG antibody (i-7) 6xHis tag (ii-7) Nickel or cobalt Methods for introducing the purification tag sequence include inserting a nucleic acid encoding the purification tag at the 5' or 3' end of the nucleic acid encoding protein (A) in an expression vector, or using a commercially available vector for introducing a purification tag.

[0029] In protein (A), the ratio of the total number of amino acids in all intervening amino acid sequences (Z) constituting protein (A), the total number of amino acids in all terminal amino acid sequences (T) constituting protein (A), and the total number of amino acids in the purification tag is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%, based on the total number of amino acids in protein (A), from the viewpoints of biocompatibility and gelation efficiency.

[0030] When protein (A) contains polypeptide chain (Y) and / or polypeptide chain (Y'), GAGAGS sequence (2), and polypeptide chain (S), from the viewpoints of biocompatibility and gelation efficiency, it is preferable that polypeptide chain (Y) or polypeptide chain (Y') and GAGAGS sequence (2) or polypeptide chain (S) are chemically bonded alternately.

[0031] The ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) is 1:0.5 to 1:20 from the viewpoints of biocompatibility and gelation efficiency. From the viewpoint of further improving the gelling efficiency, the ratio is preferably 1:1.5 to 1:20, more preferably 1:2 to 1:20, and particularly preferably 1:2 to 1:15.

[0032] Some preferred examples of the protein (A) are shown below. (A1): A protein whose amino acid sequence (X) is the GVGVP sequence (4) (A11): A protein having a polypeptide chain (Y'1) in which one amino acid in a polypeptide chain (Y1) having 2 to 200 consecutive GVGVP sequences (4) is substituted with lysine (K). (A111) A protein having a polypeptide chain (Y'1) and a polypeptide chain (S1) having 2 to 200 consecutive GAGAGS sequences (2). (A111-1): A protein having a polypeptide chain (Y'11) having an amino acid sequence (GVGVP)4GKGVP(GVGVP)3 sequence (6) shown in SEQ ID NO: 6 in which one amino acid in the polypeptide chain (Y11) of the (GVGVP)8 sequence (14) is substituted with lysine (K), which is the amino acid sequence shown in SEQ ID NO: 14 in which eight consecutive GVGVP sequences (4) are present, and a polypeptide chain (S1) having 2 to 200 consecutive GAGAGS sequences (2). (A111-1-1): A protein having a polypeptide chain (S1-1) with a (GAGAGS)4 sequence (5) in which four consecutive GAGAGS sequences (2) are shown in SEQ ID NO: 5, and a (GVGVP)4GKGVP(GVGVP)3 sequence (6). Specifically, the following proteins are included: (i) A protein (SELP8K) having a molecular mass of approximately 80 kDa and an amino acid sequence (16) shown in SEQ ID NO: 16, which has 12 (GAGAGS)4 sequences (5) and 13 (GVGVP)4GKGVP(GVGVP)3 sequences (6), which are chemically bonded alternately to a (GAGAGS)2 sequence (15) having an amino acid sequence shown in SEQ ID NO: 15. (ii) A protein (SELP8K4) having four (GAGAGS) sequences (5) and four (GVGVP)GKGVP(GVGVP) sequences (6), which are chemically bonded alternately to form an amino acid sequence (27) having a molecular mass of approximately 30 kDa and represented by SEQ ID NO: 27. (A111-1-2): A protein having a polypeptide chain (S1-2) with the (GAGAGS)2 sequence (15), which is the amino acid sequence shown in SEQ ID NO: 15, in which two consecutive GAGAGS sequences (2) are present, and a (GVGVP)4GKGVP(GVGVP)3 sequence (6). Specifically, the following proteins are included: (i) A protein (SELP0K) having a molecular mass of approximately 82 kDa and an amino acid sequence (17) represented by SEQ ID NO: 17, which has 17 copies of each of the (GAGAGS)2 sequence (15) and the (GVGVP)4GKGVP(GVGVP)3 sequence (6), which are chemically bonded alternately.

[0033] (A111-2): A protein having the amino acid sequence (GVGVP)6GKGVP(GVGVP)5 sequence (18)(Y'12) shown in SEQ ID NO: 18, in which one amino acid in a polypeptide chain of 12 consecutive GVGVP sequences (4) is substituted with lysine (K), and a polypeptide chain (S1) of 2 to 200 consecutive GAGAGS sequences (2). (A111-2-1): A protein having the (GAGAGS) sequence (19), which is the amino acid sequence shown in SEQ ID NO: 19, in which four consecutive GAGAGS sequences (2) are present, and the (GVGVP) GKGVP (GVGVP) sequence (18). Specifically, the following proteins are included: (i) A protein having an amino acid sequence (20) represented by SEQ ID NO: 20 and having a molecular mass of approximately 105 kDa, which comprises 12 (GAGAGS)4 sequences (19) and 13 (GVGVP)6GKGVP(GVGVP)5 sequences (18), which are chemically bonded alternately to one another, and a (GAGAGS)2 sequence (15) chemically bonded thereto.

[0034] (A2): A protein whose amino acid sequence (X) is the VPGVG sequence (1) (A21): A protein having a polypeptide chain (Y2) with 2 to 200 consecutive VPGVG sequences (1) and a GAGAGS sequence (2). Specifically, the following proteins are included: (i) A protein (ELP1.1) having 40 units each of the GAGAGS sequence (2), the (VPGVG) sequence (24) which is the amino acid sequence shown in SEQ ID NO: 24, and the (VPGVG) sequence (25) which is the amino acid sequence shown in SEQ ID NO: 25, and having a molecular mass of approximately 200 kDa and a structure formed by chemically bonding 40 blocks each of which is the (VPGVG) sequence (24), the GAGAGS sequence (2), and the (VPGVG) sequence (25) in this order.

[0035] (A3): A protein having a polypeptide chain (Y1) with 2 to 200 consecutive GVGVP sequences (4) and a polypeptide chain (S1) with 2 to 200 consecutive GAGAGS sequences (2). Specifically, the following proteins are included: (i) (GAGAGS)8 sequence (21), which is the amino acid sequence shown in SEQ ID NO: 21, and (GVGVP), which is the amino acid sequence shown in SEQ ID NO: 22 40 A protein (SELP6.1) having five units of each of the sequences (22), which are chemically bonded alternately, and having a molecular mass of approximately 110 kDa and an amino acid sequence (23) shown in SEQ ID NO: 23. (ii) A protein (SELP3.1) having 15 consecutive sequences of (GAGAGS)4 sequence (5) and two consecutive sequences of GVGVP sequence (4), each of which has an amino acid sequence (29) shown in SEQ ID NO: 29 and a molecular weight of 40 kDa. (iii) A protein (SELP3) having a molecular mass of approximately 90 kDa and a structure formed by alternating chemical bonds of 12 units each of the (GAGAGS)8 sequence (21) and the (GVGVP)8 sequence (14).

[0036] The protein (A) is selected from the group consisting of the protein (A111) [preferably the protein (SELP8K) of sequence (16), the protein (SELP0K) of sequence (17), the protein (20), and the protein (SELP8K4) of sequence (27)], the protein (A21) [preferably the protein (ELP1.1) of sequence (26)], and the protein (A3) [preferably the protein (SELP6.1) of sequence (23), and the protein (SELP3.1) of sequence (29)]. , protein of sequence (28) (SELP3)], and more preferably at least one protein selected from the group consisting of protein of sequence (16) (SELP8K), protein of sequence (17) (SELP0K), protein of sequence (26) (ELP1.1), protein of sequence (23) (SELP6.1), protein of sequence (29) (SELP3.1), and protein of sequence (28) (SELP3). Furthermore, protein (A) may be a protein having an amino acid sequence that is 70% or more homologous to the amino acid sequence of protein (SELP8K) of sequence (16), protein (SELP0K) of sequence (17), protein (20), protein (SELP8K4) of sequence (27), protein (ELP1.1) of sequence (26), protein (SELP6.1) of sequence (23), protein (SELP3.1) of sequence (29), or protein (SELP3) of sequence (28). Furthermore, this homology is preferably 80% or more, and more preferably 90% or more.

[0037] In the protein (A) of the present invention, the total proportion of β-turn structures and random coil structures in the protein (A) determined by circular dichroism spectroscopy is preferably 60 to 85%, more preferably 65 to 80%, and particularly preferably 70 to 75%, from the viewpoints of water solubility and gelation efficiency. Even if the protein sequence is the same, the total proportion of β-turn structures and random coil structures in the protein will vary depending on the protein production method, protein purification method, pH of the solvent in which the protein is dissolved, and the polarity of the solvent.

[0038] The method for controlling the above ratio is not particularly limited, but it can be increased or decreased by the following methods. When the above ratio is increased, for example, a dilution refolding method (large dilution method) can be used in which protein (A) alone is diluted with an excess amount of buffer solution and then refolded. In addition, when the above ratio is to be reduced, for example, a method of denaturing the protein (A) with a denaturant or heat or the like can be mentioned. The total proportion of β-turn structures and random coil structures in protein (A) is determined by the following measurement method.

[0039] <Method for measuring the total ratio of β-turn structure and random coil structure in protein (A)> The protein is dissolved in deionized water (4°C) to a concentration of 0.3 mg / ml to prepare an aqueous solution of the protein. The prepared aqueous solution of the protein is measured using a circular dichroism spectrometer (JASCO Corporation, "J-820") (measurement temperature: 4°C), and the proportions of β-turn structure and random coil structure are calculated using a secondary structure analysis program (JWSSE type: JASCO Corporation). The sum of these is taken as the total proportion of β-turn structure and random coil structure.

[0040] The molecular mass of the protein (A) as determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is preferably 15 to 200 kDa, more preferably 30 to 150 kDa, and particularly preferably 70 to 120 kDa, from the viewpoints of biocompatibility and gelation efficiency.

[0041] In the present invention, the hydrophobicity of the protein (A) is preferably 0.2 to 1.2, more preferably 0.4 to 1.0, and particularly preferably 0.42 to 0.80, from the viewpoints of water solubility, biocompatibility, and gelation efficiency. The hydrophobicity of protein (A) indicates the degree of hydrophobicity of the protein (A) molecule and can be calculated by applying the number of each amino acid residue constituting the protein (A) molecule (Mα), the hydrophobicity of each amino acid (Nα), and the total number of amino acid residues in one protein (A) molecule (MT) to the following formula: The hydrophobicity of each amino acid is determined using the following numerical values ​​found in a non-patent document (Albert L. Lehninger, David L. Nelson, Lehninger's New Biochemistry, Vol. 1, Hirokawa Shoten, September 2010, pp. 346-347): Hydrophobicity = Σ(Mα × Nα) / (MT) Mα: The number of each amino acid residue in one molecule of the artificial protein (A) Nα: Hydrophobicity of each amino acid MT: Total number of amino acid residues in one molecule of artificial protein (A) A (alanine): 1.8 R(arginine):-4.5 N (asparagine): -3.5 D (aspartic acid): -3.5 C (cysteine): 2.5 Q(glutamine):-3.5 E (glutamic acid):-3.5 G (glycine): -0.4 H (histidine): -3.2 I (Isoleucine): 4.5 L (leucine): 3.8 K (lysine): -3.9 M (methionine): 1.9 F (phenylalanine): 2.8 P(proline):-1.6 S (Serine): -0.8 T (Threonine): -0.7 W (tryptophan): -0.9 Y (tyrosine): -1.3 V(Valine): 4.2 For example, if the artificial protein (A) has the sequence (GVGVP)4GKGVP(GVGVP)3 (6), the hydrophobicity of the artificial protein (A) = {16 (number of Gs) × (-0.4) + 15 (number of Vs) × 4.2 + 8 (number of Ps) × (-1.6) + 1 (number of Ks) × (-3.9)} / 40 (total number of amino acid residues) = 1.0.

[0042] The gelled material of the present invention is derived from the protein (A) having the amino acid sequence described above, and is therefore degradable by enzymes in the living body, and therefore has excellent biodegradability.

[0043] In the present invention, protein (A) can be obtained by extraction from natural products, organic synthesis (enzymatic methods, solid-phase synthesis, liquid-phase synthesis, etc.), genetic recombination, etc. Regarding organic synthesis, methods such as those described in "Lectures on Biochemical Experiments 1, Chemistry of Proteins IV" (published July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.) and "Continued Lectures on Biochemical Experiments 2, Chemistry of Proteins (Part 2)" (published May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.) can be applied. Regarding genetic recombination, methods such as those described in Japanese Patent No. 3338441 can be applied. Although protein (A) can be obtained by extraction from natural products, organic synthesis, and genetic recombination, genetic recombination is preferred from the viewpoints of easy modification of the amino acid sequence and inexpensive mass production.

[0044] As described above, the method for producing protein (A) of the present invention is a method for producing a gel of protein (A) via step (1). The step (1) is a step of adjusting the temperature of a mixture containing the protein (A), water, and an alcohol having 1 to 4 carbon atoms to 0 to 80°C. Examples of the alcohol having 1 to 4 carbon atoms include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and tert-butyl alcohol. If the temperature is below 0°C, the gelation rate tends to be slow, and if the temperature is above 80°C, the function of the protein may be reduced. In the above steps, the temperature of the mixture is preferably 1 to 80°C, more preferably 1 to 60°C, particularly preferably 25 to 50°C, and most preferably 30 to 40°C, from the viewpoint of the thermal stability and handleability of the protein. In the step (1), when obtaining a mixture of the protein (A), water, and the alcohol having 1 to 4 carbon atoms, the order of mixing may be arbitrary, but it is preferable to obtain a mixture by dissolving the protein (A) in water and then mixing the alcohol having 1 to 4 carbon atoms. The step (1) may be carried out intermittently, and a step (2) of adjusting the temperature of the mixture containing water and the alcohol having 1 to 4 carbon atoms to less than 0°C or more than 80°C may be carried out between steps (1) and (1).

[0045] In the production method of the present invention, the time for step (1) (the total time for bringing the mixture to 0 to 80°C) is preferably 0 to 180 minutes. Note that a time for step (1) of 0 minutes means that it is less than 0.5 minutes (30 seconds). Furthermore, a time for step (1) of 0 minutes also includes an embodiment in which the protein (A) gels the moment the protein (A), water, and the alcohol having 1 to 4 carbon atoms are mixed to form a mixture at 0 to 80°C. When step (1) is carried out multiple times, the total time for step (1) is preferably 0 to 180 minutes. The total time for the step (2) is preferably 0 to 60 minutes, more preferably 0 to 30 minutes, and particularly preferably 0 minute.

[0046] Step (1) of the present invention may be considered to be completed when the mixture gels (loses fluidity). After the step (1) of the present invention is completed and the production method of the present invention is also completed, the gel may be stored at 80° C. or lower (preferably −80 to 40° C.).

[0047] The mixture may contain an inorganic salt and phosphoric acid (salt) in addition to the protein (A), water, and the alcohol having 1 to 4 carbon atoms. Examples of the inorganic salt include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, etc. In this application, phosphoric acid (salt) is not included in the inorganic salt. In this application, phosphoric acid (salt) means phosphoric acid and / or phosphate salts. Phosphoric acid (salts) include phosphoric acid and phosphate salts. Examples of the salts include alkali metal salts and alkaline earth metal salts, and specific examples include sodium salts, potassium salts, calcium salts, and magnesium salts.

[0048] From the viewpoints of biocompatibility and gelling efficiency, the weight proportion of the protein (A) contained in the mixture is preferably 5 to 45 wt %, more preferably 5 to 42 wt %, based on the weight of the mixture. From the viewpoints of biocompatibility and gelling efficiency, the weight proportion of the alcohol having 1 to 4 carbon atoms contained in the mixture is preferably 0.1 to 55 wt %, and more preferably 0.5 to 53 wt %, based on the weight of the mixture. The weight proportion of water contained in the mixture is preferably 1 to 90% by weight, more preferably 5 to 90% by weight, based on the weight of the mixture, from the viewpoint of gelling efficiency.

[0049] The gel obtained by the present invention is useful because it can be used as a tissue regeneration material. Methods for applying the gel obtained by the present invention to the affected area include a method in which a mixture containing the protein (A), water, and an alcohol having 1 to 4 carbon atoms is injected into the affected area [injected in a manner similar to the injection of an aqueous solution of protein (A) into the affected area in WO 2012 / 111438], and the temperature of the mixture is adjusted to the body temperature of the affected area, thereby performing the step (1) at the affected area and producing a gel at the affected area, and a method in which a gel obtained by performing the step (1) outside the body is applied to the affected area. [Example]

[0050] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following, parts represent parts by weight.

[0051] <Production Example 1: Production of Protein (A)> [Creating SELP8K] ○ Construction of SELP8K-producing strain Plasmid pPTS0345 encoding SELP8K was prepared according to the method described in the Examples of Japanese Patent No. 4088341. The constructed plasmid was transformed into Escherichia coli to obtain a SELP8K-producing strain. Below, we describe a method for producing SELP8K (polypeptide (A1)), a polypeptide with a molecular mass of approximately 80 kDa and a sequence (16) that contains 12 (GAGAGS)4 sequences (5), which are a type of polypeptide (A), and 13 (GVGVP)4GKGVP(GVGVP)3 sequences (6), which are chemically bonded alternately.

[0052] Cultivation of SELP8K-producing strains An overnight culture of the SELP8K-producing strain grown at 30°C was used to inoculate 50 ml of LB medium in a 250 ml flask. Kanamycin was added to the LB medium to a final concentration of 50 μg / ml to form a culture medium, and the culture medium was incubated at 30°C with stirring (200 rpm). When the turbidity of the culture medium reached OD600 = 0.8 (using a UV1700 spectrophotometer, manufactured by Shimadzu Corporation), 40 ml of the culture medium was transferred to another flask preheated to 42°C and cultured at 42°C for approximately 2 hours. The culture medium was then cooled on ice, the turbidity OD600 of the culture medium was measured, and the E. coli cells were collected by centrifugation.

[0053] Purification of SELP8K Protein was purified from the collected E. coli biomass by the following steps: Step 1: Cell lysis, Step 2: Removal of insoluble debris by centrifugation, Step 3: Ammonium sulfate precipitation, Step 4: Ultrafiltration, Step 5: Anion exchange chromatography, Step 6: Ultrafiltration, and Step 7: Lyophilization. In this manner, purified SELP8K (polypeptide (A1)) with a molecular mass of approximately 80 kDa was obtained.

[0054] Step 1: Cell lysis 200 g of deionized water was added to 100 g of collected E. coli, and the cells were lysed using a high-pressure homogenizer (55 MPa) to obtain a cell lysate containing lysed cells. The cell lysate was then adjusted to pH 4.0 with glacial acetic acid.

[0055] Step 2: Removal of insoluble debris by centrifugation The cell lysate was then centrifuged (6300 rpm, 4°C, 30 minutes) to collect the supernatant.

[0056] Step 3: Ammonium sulfate precipitation A saturated ammonium sulfate solution was added to the supernatant recovered in step 2 so that the ammonium sulfate concentration was 25% by weight. After that, the mixture was left to stand for 8 to 12 hours, and the precipitate was recovered by centrifugation. The recovered precipitate was dissolved in deionized water. Next, a saturated ammonium sulfate solution was added to the dissolved solution so that the ammonium sulfate concentration was 25% by weight. After that, the mixture was left to stand for 8 to 12 hours, and the precipitate was recovered by centrifugation. The recovered precipitate was dissolved in deionized water to obtain a solution.

[0057] Step 4: Ultrafiltration The solution obtained in step 3 was subjected to an ultrafiltration device (holofiber: manufactured by GE Healthcare) with a molecular mass cutoff of 30,000. Ultrafiltration was performed on the solution obtained in step 3 using 20 times the amount of deionized water to obtain a polypeptide solution after ultrafiltration.

[0058] Step 5: Anion Exchange Chromatography The ultrafiltrated polypeptide solution was added to 10 mM sodium acetate buffer to a polypeptide concentration of 20 g / L, and then subjected to an AKTAPrime (Amersham) equipped with an anion exchange column HiPrepSP XL16 / 10 (GE Healthcare). 500 mM sodium acetate buffer was used as the eluent, and the eluted fraction was collected.

[0059] Step 6: Ultrafiltration The eluted fraction obtained in step 5 was treated in the same manner as in "4: Ultrafiltration" above to obtain a polypeptide solution after ultrafiltration.

[0060] Step 7: Freeze drying The polypeptide solution obtained in step 6 was diluted with deionized water to a polypeptide concentration of 3 g / L, and placed in a stainless steel tray so that the water level was 10 mm or less. The solution was then placed in a freeze dryer (Nihon Techno Service Co., Ltd.) and frozen at -30°C for 24 hours. After freezing, the solution was subjected to primary drying at a vacuum of 5 Pa or less at -30°C for 110 hours, and secondary drying at a vacuum of 5 Pa or less at 30°C for 48 hours to obtain SELP8K (protein (A-1), molecular mass by SDS-PAGE: 80 kDa).

[0061] Identification of SELP8K(A-1) The protein (A) in Production Example A-1 was identified by the following procedure. Analysis was performed by Western blotting using rabbit anti-SELP8K antibody and rabbit anti-6xHis antibody (Roland) against the 6xHis tag in the C-terminal sequence. The Western blotting procedure was as follows. A band showing antibody reactivity with each antibody was observed at the apparent molecular mass of 80 kDa. In addition, Table 1 shows the amino acid composition ratio (measured value) of protein (A) related to Production Example A1 obtained by amino acid composition analysis using an amino acid analysis system (Prominence, Shimadzu Corporation) and the amino acid composition ratio (theoretical value) of SELP8K estimated from the synthetic gene sequence. From these, it was confirmed that the protein (A) of Example 1 is a protein (SELP8K) having 13 polypeptide chains (Y'2) of the (GVGVP)4GKGVP(GVGVP)3 sequence (6) in which one of the valines (V) in a polypeptide chain (Y) consisting of eight consecutive GVGVP sequences (4) has been replaced with a lysine (K), and 12 polypeptide chains (S1-1) of the (GAGAGS)4 sequence (5) in which four consecutive GAGAGS sequences (2) are chemically bonded alternately to form a sequence (16). The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A-1) was 52.9% of the total number of amino acids in the protein (A-1). Furthermore, the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A-1) (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) was 1:2.1.

[0062] [Table 1]

[0063] <Western blotting> 20 μL of Western blot sample was mixed with 10 μL of 3x SDS buffer (containing 150 mM Tris HCl (pH 6.8), 300 mM dithiothreitol, 6% sodium dodecyl sulfate (SDS), 0.3% bromophenol blue, and 30% glycerol) and heated at 95°C for 5 minutes to prepare the electrophoresis sample. SDS-PAGE was performed using 15 μL of this electrophoresis sample. After electrophoresis, the gel was transferred to a polyvinylidene fluoride membrane (hereafter referred to as "membrane"), which was then immersed in blocking buffer (containing 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween 20, and 5% skim milk) and shaken at room temperature for 1 hour to block the membrane. After blocking, the membrane was washed with TBS-T [containing 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1 wt% Tween 20] for 2 minutes. Next, the membrane was immersed in a primary antibody solution (primary antibody: anti-SELP8K antibody and anti-His-tag antibody (manufactured by Rockland) diluted 1:500 with TBS-T) and left to stand overnight at 4 ° C to allow antibody reaction. After the reaction, the membrane was washed four times with TBS-T for 5 minutes, and then immersed in a solution of a secondary antibody capable of binding to the primary antibody and conjugated with horseradish peroxidase as a labeling enzyme (secondary antibody: ECL anti-rabbit IgG HRP linked F(ab')2 fragment (manufactured by GE Healthcare) diluted 1:2000 with TBS-T) and left to stand at room temperature for 30 minutes to allow antibody reaction. After the reaction, the membrane was washed four times with TBS-T for 5 minutes each, and then subjected to an enzymatic reaction using an ECL-Advance Western Blotting Detection Kit (GE Healthcare). Bands were visualized using a Luminometer ForECL (GE Healthcare) and exposed to high-sensitivity instant black-and-white film (Fujifilm).

[0064] <Production Example 2> In the "Production of protein (A)" of Production Example 1, the procedure was repeated except that a "plasmid encoding SELP0K" was used instead of a "plasmid encoding SELP8K," to obtain SELP0K polymer (A-2) [molecular mass by SDS-PAGE: 82 kDa], which is the artificial protein (A) of sequence number (17). The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A-2) was 65.4% of the total number of amino acids in the protein (A-2). Furthermore, the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A-2) (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) was 1:4.

[0065] <Production Example 3> In the "Production of protein (A)" of Production Example 1, the procedure was repeated except that the "plasmid encoding SELP3" was used instead of the "plasmid encoding SELP8K," and the artificial protein (A) of sequence number (28), SELP3 polymer (A-3) [molecular mass by SDS-PAGE: 90 kDa], was obtained. The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A-3) was 44.1% of the total number of amino acids in the protein (A-3). Furthermore, the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A-3) (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) was 1:1.

[0066] <Production Example 4> In the "Production of protein (A)" of Production Example 1, the procedure was repeated except that the "plasmid encoding SELP8K" was replaced with a "plasmid encoding ELP1.1," to obtain ELP1.1 polymer (A-4) [molecular mass as determined by SDS-PAGE: 200 kDa], which is the artificial protein (A) of sequence number (26). The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A-4) was 89.1% of the total number of amino acids in the protein (A-4). Furthermore, the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A-4) (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) was 1:12.

[0067] <Production Example 5> In the "Production of protein (A)" of Production Example 1, the procedure was repeated except that the "plasmid encoding SELP8K" was replaced with a "plasmid encoding SELP6.1," to obtain SELP6.1 polymer (A-5) [molecular mass by SDS-PAGE: 110 kDa], which is the artificial protein (A) of sequence number (23). The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A-5) was 77.3% of the total number of amino acids in the protein (A-5). Furthermore, the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A-5) (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) was 1:5.

[0068] <Production Example 6> In the "Production of protein (A)" of Production Example 1, the procedure was repeated except that the "plasmid encoding SELP8K" was replaced with a "plasmid encoding SELP3.1," to obtain SELP3.1 polymer (A-6) [molecular mass by SDS-PAGE: 40 kDa], which is the artificial protein (A) of sequence number (29). The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A-6) was 26.6% of the total number of amino acids in the protein (A-6). Furthermore, the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A-6) (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) was 1:0.5.

[0069] <Comparative Manufacturing Example 1> In the "Production of protein (A)" of Production Example 1, the procedure was repeated except that the "plasmid encoding SELP8K" was replaced with a "plasmid encoding SLP4.1," to obtain SLP4.1 polymer (A'-1) [molecular mass by SDS-PAGE: 93 kDa], which is the artificial protein (A) of sequence number (30). SLP4.1 is a protein having 29 sequences of six consecutive GAGAGS sequences (2) and 29 sequences of two consecutive GVGVP sequences (4), which are chemically bonded alternately to form the amino acid sequence (30) shown in SEQ ID NO: 30. The total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A'-1) was 20.7% of the total number of amino acids in the protein (A'-1). Furthermore, the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A'-1) (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) was 1:0.3.

[0070] <Examples 1 to 108 and Comparative Examples 1 to 39: Production of gelled product of protein (A)> The types of proteins (A) shown in Tables 2 to 8 and deionized water at 25°C were placed in microtubes in the weights shown in Tables 2 to 8, and mixed at 37°C using a vortex mixer. Next, the components (37°C) listed in Tables 2 to 8, other than the protein (A) and deionized water, were added to the microtube in the weights listed in Tables 2 to 8 and mixed using a vortex mixer to produce a 37°C mixture to be used in the reaction. The temperature was then adjusted to 37°C, and the presence or absence of gelation was confirmed immediately after the start of temperature adjustment and every 10 minutes thereafter, according to the following criteria. (For each example, three tests were conducted: one in which ethanol was added, one in which methanol was added, and one in which 1-propanol was added. As a comparative experiment, a test in which acetic acid was added was also conducted.) The time at which gelation was first confirmed (the time elapsed from the start of temperature control until gelation was first confirmed) is shown in Tables 2 to 8 (if gelation was not confirmed even after 190 minutes, it was marked as ×). The occurrence of gelation was judged according to the following criteria. Gelling: A microtube containing the mixture was turned upside down, and if the solution did not drip, it was determined that gelation had occurred. Non-gelling: When the microtube containing the mixture was turned over, if the solution dripped, it was determined that the mixture had not gelled.

[0071] <Examples 109 to 114 and Comparative Example 40: Production of gelled product of protein (A)> The type of protein (A) shown in Table 9 and deionized water at 25°C were placed in a microtube in the weights shown in Table 9, and mixed at 1°C using a vortex mixer. Next, the components (1°C) listed in Table 9, other than the protein (A) and deionized water, were added to the microtube in the weights listed in Table 9 and mixed using a vortex mixer to produce a 1°C mixture to be used in the reaction. The temperature was then adjusted to 1°C, and the presence or absence of gelation was confirmed immediately after the start of temperature adjustment and every 10 minutes thereafter using the same criteria as in Example 1. (For each Example, three tests were conducted: one in which ethanol was added, one in which methanol was added, and one in which 1-propanol was added. As a comparative experiment, a test in which acetic acid was added was also conducted.) The time at which gelation was first confirmed (the time elapsed from the start of temperature control to the time at which gelation was first confirmed) is shown in Table 9 (if gelation was not confirmed even after 190 minutes, it was marked as ×).

[0072] <Examples 115 to 120 and Comparative Example 41: Production of gelled product of protein (A)> The type of protein (A) shown in Table 10 and deionized water at 25°C were placed in a microtube in the weights shown in Table 10, and mixed using a vortex mixer at 60°C. Next, the components (60°C) listed in Table 10, other than the protein (A) and deionized water, were added to the microtube in the weights listed in Table 10 and mixed using a vortex mixer to produce a 60°C mixture to be used in the reaction. The temperature was then adjusted to 60°C, and the presence or absence of gelation was confirmed immediately after the start of temperature adjustment and every 10 minutes thereafter using the same criteria as in Example 1. (For each Example, three tests were conducted: one in which ethanol was added, one in which methanol was added, and one in which 1-propanol was added. As a comparative experiment, a test in which acetic acid was added was also conducted.) Table 10 shows the time when gelation was first confirmed (the time elapsed from the start of temperature control until gelation was first confirmed).

[0073] [Table 2]

[0074] [Table 3]

[0075] [Table 4]

[0076] [Table 5]

[0077] [Table 6]

[0078] [Table 7]

[0079] [Table 8]

[0080] [Table 9]

[0081] [Table 10]

[0082] It can be seen that Examples 1 to 120, in which an alcohol having 1 to 4 carbon atoms (methanol, ethanol, or 1-propanol) was added, showed a dramatic improvement in gelation rate compared to Comparative Examples 1 to 18 and 40 to 41, in which no such compound was added. Furthermore, Comparative Examples 19 to 36, in which the ratio of "GAGAGS sequence (2):total of amino acid sequence (X) and amino acid sequence (X')" is outside the range of "1:0.5 to 1:20," also show an increased gelation rate compared to Comparative Examples 37 to 39, in which no alcohol having 1 to 4 carbon atoms is added. On the other hand, it can be seen that the degree of increase in gelation rate in Examples 1 to 120, in which the ratio is within the range of "1:0.5 to 1:20" (the increase from Comparative Examples 1 to 18 and 40 to 41, in which no such compound is added), is significantly greater than the degree of increase in Comparative Examples 19 to 36. [Industrial Applicability]

[0083] The production method of the present invention is useful because it can gelatinize the protein (A) in a short time and the gel can be used as a tissue regeneration material.

Claims

1. A method for producing a gelled product, in which a gelled product of protein (A) is obtained through step (1), comprising the steps of: the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and the polypeptide chains (Y') in the protein (A) is 1 to 100; the polypeptide chain (Y) is a polypeptide chain having 2 to 200 consecutive amino acids of at least one amino acid sequence (X) selected from the amino acid sequence VPGVG (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP (4) shown in SEQ ID NO: 4, and the amino acid sequence GAHGPAGPK (3) shown in SEQ ID NO: 3; the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of the lysines and arginines is 1 to 100; the total number of amino acids constituting the amino acid sequence (X) and the amino acid sequence (X') contained in the protein (A) is 20 to 90% based on the total number of amino acids in the protein (A); the amino acid sequence (X') is an amino acid sequence in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine, the protein (A) has a GAGAGS sequence (2), which is the amino acid sequence shown in SEQ ID NO: 2, and the ratio of the number of the GAGAGS sequence (2) to the total number of the amino acid sequence (X) and the amino acid sequence (X') in one molecule of the protein (A) (GAGAGS sequence (2):total of the amino acid sequence (X) and the amino acid sequence (X')) is 1:0.5 to 1:20; The production method, wherein the step (1) is a step of adjusting the temperature of a mixture containing the protein (A), water, and an alcohol having 1 to 4 carbon atoms to 0 to 80°C.

2. The method according to claim 1, wherein the protein (A) is at least one protein selected from the group consisting of a protein (A111) having a polypeptide chain (Y'1) in which one amino acid in the amino acid sequence of 2 to 200 consecutive amino acids of the GVGVP sequence (4) is substituted with lysine and a polypeptide chain (S1) of 2 to 200 consecutive amino acids of the GAGAGS sequence (2); a protein (A21) having a polypeptide chain (Y2) of 2 to 200 consecutive amino acids of the VPGVG sequence (1) and the GAGAGS sequence (2); and a protein (A3) having a polypeptide chain (Y1) of 2 to 200 consecutive amino acids of the GVGVP sequence (4) and a polypeptide chain (S1) of 2 to 200 consecutive amino acids of the GAGAGS sequence (2).

3. The protein (A111) has the amino acid sequence shown in SEQ ID NO: 6 (GVGVP). ４ GKGVP (GVGVP) ３ The method according to claim 2, wherein the protein (A111-1) has a polypeptide chain (Y'11) having the sequence (6) and a polypeptide chain (S1) having 2 to 200 consecutive GAGAGS sequences (2).

4. The method according to any one of claims 1 to 3, wherein the molecular mass of the protein (A) determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is 15 to 200 kDa.

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