Block copolymer, method for producing block copolymer, and artificial protein

Abstract

This block copolymer has a first segment containing a protein structure, and one or more second segments containing a molecular group that has a function of plasticizing the protein structure and is bonded to the first segment. The protein structure includes a main sequence and hydrophobic tags bonded to the N-terminus and the C-terminus of the main sequence, and has at least two points of reaction for reacting with the second segment and a compound having a reactive group. Among the points of reaction, the point of reaction that is on the most N-terminus side: is present in the hydrophobic tag on the N-terminus side; is the NH2 which is the N-terminus of the protein structure; or is present in the main sequence. Among the points of reaction, the point of reaction on the most C-terminus side is present in the hydrophobic tag on the C-terminus side or is present within 9 residues from the C-terminus of the main sequence.

Description

Block copolymer, method for producing block copolymer, and artificial protein

[0001] The present invention relates to a block copolymer, a method for producing a block copolymer, and an artificial protein.

[0002] Currently, proteins or block copolymers formed by binding proteins with other structures are industrially used in various fields. For example, proteins are bound to molecules with plasticizing properties to form block copolymers, which impart flexibility and other properties.

[0003] For example, Patent Document 1 describes a synthetic polymer having a first segment including a polypeptide backbone and one or more second segments bound to the first segment, wherein the second segment includes a molecular group having a plasticizing function for the polypeptide backbone.

[0004] International Publication No. 2021 / 187502

[0005] In recent years, block copolymer technology has attracted significant attention due to its versatile applications in various industries, including film manufacturing, leather processing, and textile manufacturing. At the same time, the use of structural proteins, such as fibroin, as an alternative to traditional structural materials has emerged as a notable trend, primarily due to their exceptional durability and robustness. Furthermore, as described in U.S. Patent Publication No. 2009 / 0129994, there has been growing interest in developing fibroin-containing block copolymer materials and improving their overall properties to address the inherent advantages of fibroin. However, despite the growing demand, the production of fibroin-based block copolymer materials faces challenges, including low productivity and poor reactivity between fibroin and other polymer components essential for block copolymer synthesis. Furthermore, the manufacturing process itself is characterized by complexity, making effective mass production difficult. Addressing these challenges is crucial for enabling efficient mass production and realizing the full potential of these advanced materials. The inventors focused on these challenges and investigated ways to improve the manufacturing process for fibroin-containing block copolymers, resulting in the completion of the present invention.

[0006] An object of the present invention is to provide a block copolymer with excellent productivity, a method for producing the same, and an artificial protein with excellent productivity for producing a block copolymer.

[0007] Representative embodiments of the present invention are described below.

[0008] <1> A block copolymer having a first segment containing a protein structure and one or more second segments containing a molecular group having a plasticizing function for the protein structure and bound to the first segment, wherein the protein structure includes a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and has at least two reactive sites that react with the second segment and a compound having a reactive group, and the reactive site closest to the N-terminus of the reactive sites is present within the hydrophobic tag on the N-terminus side or is present between the N-terminus of the protein structure and an NH ２or is present in the main sequence, and has a protein structure in which the most C-terminal reactive site among the reactive sites is present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. <2> The block copolymer according to <1>, in which the most C-terminal reactive site among the reactive sites is present at the C-terminus of the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. <3> The block copolymer according to <1> or <2>, in which the most N-terminal reactive site among the reactive sites is present at the N-terminus of the N-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. <4> The block copolymer according to any of <1> to <3>, in which the most N-terminal reactive site present in the main sequence and the most C-terminal reactive site present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence are thiol groups. <5> The block copolymer according to <4>, in which the compound has a thiol-reactive group that reacts with the thiol. <6> The block copolymer according to any one of <1> to <3>, wherein, of the reactive sites, the most N-terminal reactive site present in the N-terminal hydrophobic tag and the most C-terminal reactive site present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence are amino groups. <7> The block copolymer according to <6>, wherein the compound has an amino-reactive group that reacts with the amine. <8> The block copolymer according to <1> to <7>, wherein the hydrophobic tag is GFIL or two or more repeats of GFIL. <9> The block copolymer according to any one of <1> to <8>, wherein the molecular weight of the protein structure is 1 to 300 kDa. <10> A method for producing a block copolymer by bonding a compound having a first segment including a protein structure with a compound having a second segment containing a molecular group having a plasticizing function for the protein structure, wherein the protein structure includes a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and has at least two reactive sites that react with the second segment and a compound having a reactive group, and the reactive site closest to the N-terminus of the reactive sites is present within the hydrophobic tag on the N-terminus side or is present at the N-terminus of the protein structure. ２or is present in a main sequence, and the most C-terminal reactive site among the reactive sites is present in a C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. <11> A method for producing a block copolymer according to <10>, comprising a step of bonding a compound having the first segment and a compound having the second segment by mechanochemical treatment. <12> A method for producing a block copolymer according to <10>, comprising adding a compound having the first segment and a compound having the second segment to a solvent, wherein the compound having the second segment is added in a non-solution state. <13> A method for producing a block copolymer according to <12>, comprising adding a reducing agent, a compound having the first segment, and a compound having the second segment to the solvent in this order. <14> An artificial protein comprising a main sequence and hydrophobic tags attached to the N-terminus and C-terminus of the main sequence, and comprising the hydrophobic tags at the N-terminus and C-terminus, and having at least two cysteine ​​residues, wherein the N-terminal most cysteine ​​residue of the cysteine ​​residues is present in the main sequence, and the C-terminal most cysteine ​​residue of the cysteine ​​residues is present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. <15> An artificial protein comprising a main sequence and hydrophobic tags attached to the N-terminus and C-terminus of the main sequence, and comprising hydrophobic tags at the N-terminus and C-terminus, and having at least one lysine residue, wherein the N-terminal most lysine residue of the lysine residues is present in the N-terminal hydrophobic tag or there is no lysine residue in the N-terminal hydrophobic tag, and the C-terminal most lysine residue of the lysine residues is present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. <16> The artificial protein according to <14> or <15>, wherein the hydrophobic tag is GFIL or two or more repeats of GFIL. <17> The artificial protein according to any one of <14> to <16>, wherein the molecular weight is 1 to 300 kDa.

[0009] According to the present invention, there are provided a block copolymer with excellent productivity, a method for producing the same, and an artificial protein with excellent productivity for producing a block copolymer.

[0010] 1 is a diagram showing experimental results in Example 1. FIG. 2 is a diagram showing experimental results in Example 2. FIG. 3 is a diagram showing experimental results in Example 2. FIG. 4 is a diagram showing experimental results in Example 3. FIG. 5 is a diagram showing experimental results in Example 4.

[0011] The main embodiments of the present invention will be described below. However, the present invention is not limited to the explicitly described embodiments. In this specification, a numerical range expressed using the symbol "to" means a range including the numerical values ​​before and after "to" as the lower and upper limits, respectively. In this specification, the term "process" includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended effect of the process is achieved. In this specification, unless otherwise specified, the temperature is 23°C, the pressure is 101,325 Pa (1 atmosphere), and the humidity (relative humidity) is 50% RH. In this specification, a combination of preferred aspects is a more preferred aspect.

[0012] (Block Copolymer) The block copolymer according to this embodiment is a block copolymer having a first segment containing a protein structure and one or more second segments containing a molecular group having a plasticizing function for the protein structure and bound to the first segment, wherein the protein structure includes a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and has at least two reactive sites that react with the second segment and a compound having a reactive group, and the reactive site closest to the N-terminus of the reactive sites is present within the hydrophobic tag on the N-terminus side or is present at the NH ２ or is present within the main sequence, and the reactive site closest to the C-terminus of the reactive sites is present within the hydrophobic tag on the C-terminus side or is a protein structure present within 9 residues from the C-terminus of the main sequence.

[0013] The block copolymer of the present invention has excellent productivity. The productivity of the block copolymer here can also be said to be the reactivity of the first segment with the second segment and a compound having a reactive group. Specifically, the protein structure includes hydrophobic tags at both ends, and the most N-terminal reactive site is present within the N-terminal hydrophobic tag or at the N-terminal NH ２ or its presence within the main sequence, the reactive site is more likely to be exposed on the surface, enhancing reactivity with the compound, and as a result, it is believed that productivity of the block copolymer as a reactant is improved. Furthermore, if the reactive site is one capable of binding protein structures together, such as a thiol group, it is believed that binding of protein structures together at the reactive site is suppressed, thereby enhancing reactivity with the compound. Furthermore, if the most C-terminal reactive site is present within the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence, it is believed that the reactive site is more likely to be exposed on the surface, resulting in excellent reactivity with the compound. When the most C-terminal reactive site is present within 9 residues from the C-terminus of the main sequence, in order to enhance reactivity with the compound, the reactive site may be present within 8 residues, 7 residues, 6 residues, 5 residues, 4 residues, 3 residues, 2 residues, or the C-terminus of the main sequence. Furthermore, even when the reactive site closest to the N-terminus is present within the main sequence, in order to enhance the reactivity with the compound, such reactive site may be present within 9 residues, 8 residues, 7 residues, 6 residues, 5 residues, 4 residues, 3 residues, 2 residues, or at the N-terminus of the main sequence from the N-terminus. In addition, when the reactive site in the protein structure contained in the block copolymer according to this embodiment is a thiol group present at the above-mentioned position, there is a particular advantage in that the productivity of the protein structure is also enhanced.

[0014] <First Segment> [Protein Structure] The protein structure includes a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence. Such a protein structure may be chemically modified, for example, by acylation of a hydroxyl group of a serine, threonine, or tyrosine residue, an amino group of a lysine residue, or the like, or a sulfhydryl group of a cysteine ​​residue. A protein structure chemically modified by such acylation or the like can have an electrophilic functional group introduced therein, thereby imparting various functionalities to the protein structure.

[0015] [Hydrophobic Tag] A hydrophobic tag refers to a tag sequence formed from hydrophobic amino acid residues. However, the C-terminus of the hydrophobic tag may be an amino acid residue containing the above-mentioned reactive site (e.g., a cysteine ​​residue or a lysine residue), and the N-terminus of the hydrophobic tag may be an amino acid residue containing the above-mentioned reactive site (e.g., a lysine residue). Here, hydrophobic amino acids refer to alanine (A), valine (V), glycine (G), isoleucine (I), leucine (L), phenylalanine (F), proline (P), tryptophan (W), and tyrosine (Y). That is, in the present disclosure, a hydrophobic tag refers to a tag sequence formed solely from hydrophobic amino acid residues, which is a region consisting of only consecutive hydrophobic amino acid residues from the terminal amino acid residue (however, if the terminal has a reactive site such as a cysteine ​​residue or a lysine residue), up to the first non-hydrophobic amino acid residue (an amino acid residue other than the hydrophobic amino acid) found.

[0016] Among these, the hydrophobic tag is preferably GFIL (glycine, phenylalanine, isoleucine, leucine) or two or more repeats of GFIL, more preferably two or more repeats of GFIL, and even more preferably GFILGFIL. In the above-mentioned aspect, the N-terminus or C-terminus of the hydrophobic tag may be an amino acid residue containing the above-mentioned reactive site (e.g., a cysteine ​​residue or a lysine residue).

[0017] [Main Sequence] The main sequence of a protein structure is preferably a sequence consisting of an artificial protein. Here, artificial proteins include recombinant proteins and synthetic proteins. In other words, as used herein, "artificial protein" refers to a protein produced artificially. An artificial protein may have a domain sequence that is different from the amino acid sequence of a naturally occurring protein, or it may have a domain sequence that is identical to the amino acid sequence of a naturally occurring protein. Furthermore, an "artificial protein" may use the amino acid sequence of a naturally occurring protein as is, or may be a protein whose amino acid sequence has been modified based on the amino acid sequence of a naturally occurring protein (e.g., a protein whose amino acid sequence has been modified by modifying the gene sequence of a cloned naturally occurring protein), or may be a protein that has been artificially designed and synthesized without relying on a naturally occurring protein (e.g., a protein having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding a designed amino acid sequence).

[0018] Furthermore, examples of artificial proteins include proteins that can be used for industrial purposes. "Usable for industrial purposes" means that the protein can be used, for example, in various general-purpose materials for indoor and outdoor use. Specific examples of artificial proteins that can be used for industrial purposes include artificial structural proteins.

[0019] [Artificial structural proteins] Structural proteins refer to proteins involved in the structure of living organisms, proteins that constitute structures produced by living organisms, or proteins derived therefrom. Artificial structural proteins also refer to proteins that self-aggregate under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. Furthermore, artificial structural proteins can also be said to be proteins that contain repeated motifs consisting of a characteristic amino acid sequence or a specific number of amino acid residues and form the skeleton of an organism or material. Artificial structural proteins are artificially produced versions of these structural proteins. Examples of such artificial structural proteins include artificial fibroin, artificial keratin, artificial collagen, artificial elastin, and artificial resilin.

[0020] When forming artificial structural proteins, amino acids with relatively small side chains are more likely to form hydrogen bonds and thus produce stronger molded products. Furthermore, alanine and glycine residues are amino acids with nonpolar side chains, and therefore are arranged so that they face inward during the folding process of protein production, making them more likely to form α-helix or β-sheet structures. Therefore, a high proportion of amino acids such as glycine and alanine residues is desirable. From the viewpoint of obtaining molded products with superior strength, the alanine residue content may be, for example, 10-40%, and may be 12-40%, 15-40%, 18-40%, 20-40%, or 22-40%. From the viewpoint of obtaining molded products with superior strength, the glycine residue content may be, for example, 10-55%, and may be 11-55%, 13-55%, 15-55%, 18-55%, 20-55%, 22-55%, or 25-55%.

[0021] As used herein, the term "alanine residue content" refers to the number of alanine residues relative to the total number of amino acid residues constituting a protein, and is a value expressed by the following formula:

[0022] Alanine residue content = (number of alanine residues contained in protein / total number of amino acid residues in protein) x 100 (%)

[0023] Furthermore, the glycine residue content, serine residue content, threonine residue content, proline residue content, and tyrosine residue content have the same meaning as those obtained by replacing the alanine residue in the above formula with glycine residue, serine residue, threonine residue, proline residue, and tyrosine residue, respectively.

[0024] It is preferable that the artificial structural protein contains amino acids with relatively large side chains or flexible amino acids uniformly throughout its entire sequence to a certain extent. Specifically, the structural protein may contain a motif containing tyrosine, threonine, and proline residues in a repeated cycle. Such a structural protein is likely to inhibit the formation of strong intermolecular hydrogen bonds during processing of the molded article obtained by molding, thereby improving processability. For example, the total content of proline, threonine, and tyrosine residues in any 20 consecutive amino acid residues may be 5% or more, more than 5.5%, 6.0% or more, more than 6.5%, 7.0% or more, more than 7.5%, 8.0% or more, more than 8.5%, 9.0% or more, 10.0% or more, or 15.0% or more. Furthermore, for example, the total content of proline, threonine, and tyrosine residues in any 20 consecutive amino acid residues may be 50% or less, 40% or less, 30% or less, or 20% or less.

[0025] The artificial structural protein may have an amino acid sequence that includes a repeat sequence. That is, the artificial structural protein according to this embodiment may have multiple amino acid sequences (repeat sequence units) with high sequence identity within the artificial structural protein. The number of amino acid residues in the repeat sequence unit is preferably 6 to 200. Furthermore, the sequence identity between the repeat sequence units may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The hydrophobicity (average hydropathic index) of the repeating sequence unit may be, for example, -0.80 or more, -0.70 or more, -0.60 or more, -0.50 or more, -0.40 or more, -0.30 or more, -0.20 or more, -0.10 or more, 0.00 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more. The upper limit of the hydrophobicity of the repeating sequence unit is not particularly limited, but may be, for example, 1.0 or less or 0.7 or less.

[0026] The artificial structural protein is (A) ｎThe amino acid sequence may include an amino acid sequence containing the motif. ｎ The motif refers to an amino acid sequence that is mainly composed of alanine residues. (A) ｎ The number of amino acid residues in the motif may be 2 to 27, or may be an integer of 2 to 20, 2 to 16, or 2 to 12. ｎ The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the motif is composed of only alanine residues). ｎ Preferably, the artificial structural protein contains a plurality of motifs. ｎ When there are multiple motifs, their (A) ｎ The motifs may have the same amino acid sequence or different amino acid sequences. ｎ The amino acid sequence containing the motif is preferably different from the amino acid sequence of a naturally occurring protein. ｎ The amino acid sequence of the present invention is different from the amino acid sequence of a naturally occurring protein, and (A) ｎ The motif represents an amino acid sequence consisting of 2 to 27 amino acid residues, and (A) ｎ In another preferred embodiment of the present invention, the number of alanine residues in the motif is 40% or more of the total number of amino acid residues. ｎ Preferred embodiments of the motif are as described above.

[0027] The artificial structural protein may be artificial fibroin. Examples of fibroin include naturally occurring fibroin. Examples of naturally occurring fibroin include fibroin produced by insects or spiders. Natural fibroin is a fibrous protein with a molecular weight of approximately 370,000, composed of two subunits, and has a high content of glycine, alanine, serine, and tyrosine residues, with these amino acid residues accounting for nearly 90% of the total number of amino acid residues. Natural fibroin has a crystalline region rich in amino acid residues with relatively small side chains such as glycine, alanine, and serine, and an amorphous region containing amino acid residues with relatively large side chains such as tyrosine.

[0028] More specific examples of naturally occurring fibroins include fibroins whose sequence information is registered in NCBI GenBank. For example, among the sequences registered in NCBI GenBank that contain INV as a division, the sequence can be confirmed by extracting sequences in which spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" are described as keywords in the DEFINITION, a specific product character string from the CDS, or a specific character string in the TISSUE TYPE from the SOURCE.

[0029] As used herein, "artificial fibroin" refers to artificially produced fibroin (artificial fibroin). The artificial fibroin may be a fibroin with an amino acid sequence different from that of naturally occurring fibroin, or may be a fibroin with the same amino acid sequence as that of naturally occurring fibroin. Artificial fibroin can be produced by known methods, for example, by the method described in International Publication No. 2019 / 194263. When the modified fibroin is expressed by forming an insoluble body within cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the modified fibroin as a precipitate fraction. The recovered insoluble body of the modified fibroin may be inactivated by adding citric acid and water (e.g., at 80°C for 2 hours), washed with water by filter press filtration, granulated, and dried to obtain a purified sample.

[0030] Artificial fibroin may be a fibrous protein having a structure similar to that of naturally occurring fibroin, or may be a fibroin having a sequence similar to the repetitive sequence of naturally occurring fibroin. The "similar sequence to the repetitive sequence of fibroin" may be a sequence actually found in naturally occurring fibroin, or a sequence similar thereto.

[0031] "Artificial fibroin" may be a naturally occurring fibroin whose amino acid sequence has been modified (e.g., an amino acid sequence modified by modifying the gene sequence of a cloned naturally occurring fibroin), as long as it has the amino acid sequence specified in this disclosure. Alternatively, it may be an artificially designed amino acid sequence independent of naturally occurring fibroin (e.g., an artificial fibroin having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding a designed amino acid sequence). Artificial fibroins whose amino acid sequence has been modified are also included in the category of artificial fibroin, provided that the amino acid sequence differs from that of naturally occurring fibroin. Examples of artificial fibroins include artificial silk fibroin (a silk protein produced by silkworms) and artificial spider silk fibroin (a spider silk protein produced by spiders) whose amino acid sequence has been modified. Because artificial fibroin is relatively easy to fibrillate and has high fiber-forming ability, it is preferable for the molding material to contain, and more preferably consist of, artificial spider silk fibroin.

[0032] Artificial fibroin has the formula 1: [(A) ｎ motif-REP] ｍ , or Formula 2: [(A) ｎ motif-REP] ｍ -(A) ｎ The artificial fibroin may be a protein containing a domain sequence represented by a motif. The artificial fibroin may have further amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but are not limited to, regions that do not have repeats of the amino acid motif characteristic of fibroin and consist of about 100 amino acid residues.

[0033] As used herein, a "domain sequence" refers to a sequence of a molecule having the formula 1: [(A) ｎ motif-REP] ｍ , or Formula 2: [(A) ｎ motif-REP] ｍ -(A) ｎThe amino acid sequence represented by the motif (A) ｎ The motif represents an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues is an integer between 2 and 27. (A) ｎ The number of amino acid residues in the motif may be an integer of 2 to 27, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. ｎ The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the motif is composed of only alanine residues). ｎ At least seven of the motifs may be composed of only alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may also be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. (A) ｎ The motifs may have the same or different amino acid sequences, and the multiple REPs may have the same or different amino acid sequences.

[0034] Specific examples of artificial fibroins include, for example, an artificial fibroin derived from a major spinal dragline silk protein produced in the major ampullate gland of spiders (first artificial fibroin) as described in WO 2019 / 194263, an artificial fibroin having a domain sequence with a reduced content of glycine residues (second artificial fibroin), (A) ｎ An artificial fibroin having a domain sequence with a reduced motif content (third artificial fibroin), a glycine residue content, and (A) ｎThese include an artificial fibroin with a reduced motif content (fourth artificial fibroin), an artificial fibroin with a domain sequence containing a region with a locally high hydrophobic index (fifth artificial fibroin), and an artificial fibroin with a domain sequence with a reduced content of glutamine residues (sixth artificial fibroin). The definitions of the first to sixth artificial fibroins are set forth in WO 2019 / 194263, the contents of which are incorporated herein by reference.

[0035] The artificial fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus, which allows the artificial fibroin to be isolated, immobilized, detected, visualized, etc. The artificial fibroin may also have a hinge sequence between the N-terminus of the main sequence and the hydrophobic tag on the N-terminus side.

[0036] An example of a tag sequence is an affinity tag that utilizes specific affinity (binding property, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). A His tag is a short peptide consisting of approximately 4 to 10 histidine residues, and has the property of specifically binding to metal ions such as nickel, so it can be used to isolate artificial fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 8 (an amino acid sequence including a His tag sequence and a hinge sequence).

[0037] Furthermore, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione, and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

[0038] Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody specific to the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag. By using an epitope tag, artificial fibroin can be easily purified with high specificity.

[0039] Furthermore, a tag sequence that can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with the protease, the artificial fibroin from which the tag sequence has been cleaved can be recovered.

[0040] Specific examples of artificial fibroins include those represented by SEQ ID NOS: 1 to 7. The artificial fibroin may be an artificial fibroin represented by SEQ ID NOS: 1 to 7 or an artificial fibroin containing an amino acid sequence having 90% or more sequence identity with these amino acid sequences. The respective contents of alanine residues, glycine residues, serine residues, threonine residues, tyrosine residues, glutamine residues, and lysine residues in the artificial fibroins represented by SEQ ID NOS: 1 to 7 are shown in Table 1 below. The artificial fibroins represented by SEQ ID NOS: 1 and 7 correspond to the aforementioned fourth artificial fibroin, the artificial fibroins represented by SEQ ID NOS: 2, 3, 5, and 6 correspond to the aforementioned sixth artificial fibroin, and the artificial fibroin represented by SEQ ID NOS: 4 corresponds to the aforementioned first artificial fibroin.

[0041]

[0042] The artificial fibroin may be an artificial fibroin having at least two or more characteristics of the first artificial fibroin, the second artificial fibroin, the third artificial fibroin, the fourth artificial fibroin, the fifth artificial fibroin, and the sixth artificial fibroin.

[0043] The molecular weight of the artificial fibroin according to this embodiment is not particularly limited, and may be, for example, 2 kDa or more and 700 kDa or less. The molecular weight of the artificial fibroin according to this embodiment may be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, or 700 kDa or less, 600 kDa or less, 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less.

[0044] [Reactive Points] The protein structure has at least two reactive points that react with the second segment and the compound having a reactive group. In the block copolymer, the reactive points are in the structure after reaction with the compound and serve as bonding points with the second segment.

[0045] The reactive site may be, for example, at least one selected from the group consisting of a thiol group, an amino group, a hydroxy group, a guanidino group, a carboxy group, a phenoxy group, an indole group, an amide group, an azide group, and an alkynyl group, preferably at least one selected from the group consisting of a thiol group, an amino group, a hydroxy group, a guanidino group, a carboxy group, a phenoxy group, an indole group, and an amide group, more preferably at least one selected from the group consisting of a thiol group, an amino group, a hydroxy group, a guanidino group, a carboxy group, a phenoxy group, and an indole group, and even more preferably at least one selected from the group consisting of a thiol group, an amino group, a hydroxy group, a guanidino group, a carboxy group, a phenoxy group, and an indole group. The amino group may be at least one selected from the group consisting of a phenoxy group, a hydroxyl group, a guanidino group, and a carboxyl group, more preferably at least one selected from the group consisting of a thiol group, an amino group, a hydroxyl group, a guanidino group, and a carboxyl group, even more preferably at least one selected from the group consisting of a thiol group, an amino group, a hydroxyl group, and a guanidino group, particularly preferably at least one selected from the group consisting of a thiol group, an amino group, and a hydroxyl group, particularly preferably at least one selected from the group consisting of a thiol group and an amino group, and most preferably a thiol group. The amino group may be, for example, the amino group contained in lysine. The hydroxy group may be, for example, the hydroxyl group contained in serine, the hydroxyl group contained in threonine, etc. The guanidino group may be, for example, the guanidino group contained in arginine. The carboxy group may be, for example, the carboxyl group contained in glutamic acid, the carboxyl group contained in aspartic acid, etc. The phenoxy group may be, for example, the phenoxy group possessed by tyrosine. The indole group may be, for example, the indole group possessed by tryptophan. The amide group may be, for example, the amide group possessed by glutamine, the amide group possessed by asparagine, etc. The alkynyl group may be an alkynyl group introduced by reacting a thiol group possessed by cysteine ​​with an acetylene halide.

[0046] The number of reactive sites in the protein structure is preferably 2 to 5, more preferably 2 to 4, even more preferably 2 or 3, and particularly preferably 2.

[0047] The most N-terminal reactive site of the reactive sites is present in the main sequence or at the N-terminus of the protein structure, NH ２ The most N-terminal reactive site among the reactive sites is either present in the N-terminal hydrophobic tag or NH ２ or may be present within 9 residues from the end of the main sequence (i.e., the binding point with the N-terminal hydrophobic tag), may be present within the N-terminal hydrophobic tag, or may be present at the N-terminus of the protein structure, NH ２ or may be present within 8 residues, be present in the N-terminal hydrophobic tag, or be present at the N-terminus of the protein structure, NH ２ or may be present within 7 residues, be present in the N-terminal hydrophobic tag, or be present at the N-terminus of the protein structure, NH ２ or may be present within 6 residues, be present in the N-terminal hydrophobic tag, or be present at the N-terminus of the protein structure, NH ２ or may be present within 5 residues, and may be present within the N-terminal hydrophobic tag or at the N-terminus. ２ or may be present within four residues, be present in the N-terminal hydrophobic tag, or be present at the N-terminus of the protein structure, NH ２ or may be present within 3 residues, be present in the N-terminal hydrophobic tag, or be present at the N-terminus of the protein structure, NH ２ or may be present within two residues, be present in the N-terminal hydrophobic tag, or be present at the N-terminus of the protein structure, NH ２Alternatively, it may be present at the N-terminus of the main sequence. When a hinge sequence is present between the N-terminus of the main sequence and the hydrophobic tag, it is considered that by configuring the hinge sequence with amino acids having few side chains, it is possible to advantageously prevent the side chains of the amino acids constituting the hydrophobic tag from inhibiting binding with the compound at the reactive site present at the N-terminus of the main sequence.

[0048] The most C-terminal reactive site among the reactive sites is present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. Here, in one preferred embodiment of the present invention, the most C-terminal reactive site among the reactive sites is present at the C-terminus of the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. Here, from the viewpoint of the maximum elongation when a fiber, film, or the like is formed from the block copolymer, the reactive point closest to the C-terminus among the reactive points may be present within the C-terminal hydrophobic tag, or may be present within 9 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present within 8 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present within 7 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present within 6 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present within 5 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present within 4 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present within 3 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present within 2 residues from the C-terminus of the main sequence, or may be present within the C-terminal hydrophobic tag, or may be present at the C-terminus of the main sequence. In another preferred embodiment of the present invention, the reactive site is located at the C-terminus of the hydrophobic tag. When the most N-terminal reactive site in a protein structure is located within the main sequence and the most C-terminal reactive site is located within the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence, both of these reactive sites may be composed of thiol groups. When such a protein structure is, for example, an artificial protein (recombinant protein) produced by forming an inclusion body in a host cell, improved productivity of the protein structure (recombinant protein) is expected.Although the reason for this is not clear, it is thought that the protein structure has hydrophobic tags on the N- and C-terminal sides of the main sequence, which results in larger inclusion bodies formed in host cells and an increased recovery rate of the recombinant protein from the host cells, and furthermore, the presence of thiol groups at the above positions allows them to be spaced apart. Note that, when the most N-terminal reactive site in the protein structure is present within the hydrophobic tag and the most C-terminal reactive site is present within 9 residues from the C-terminus of the main sequence, for example, both of these reactive sites may be composed of amino groups, and the N-terminal reactive site may be present at the N-terminus of the hydrophobic tag. Specifically, a block copolymer having such a protein structure has the advantage that the bond between the first segment and the second segment and the compound having a reactive group is stably maintained for a longer period of time. When the N-terminal reactive point and the C-terminal reactive point are amino groups, for example, a thiol group (which does not function as a reactive point in this embodiment) may be present within 9 residues from the N-terminus of the main sequence (within 9 residues excluding the amino group if an amino group is present at the N-terminus), and may also be present at the C-terminus of the hydrophobic tag on the C-terminus. In such a protein structure, reactivity with the compound is enhanced, which can increase the productivity of the block copolymer, and for the reasons described above, improved productivity of the protein structure itself is also expected. When the N-terminal reactive point and the C-terminal reactive point are amino groups, the most N-terminal reactive point is NH, which is the N-terminus of the protein structure. ２ It is also preferable that the most C-terminal reactive site is present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. ２ is, for example, the NH of the terminal methionine encoded by the start codon AUG. ２ Alternatively, when the protein structure is modified, such as when methionine is cleaved by methionyl aminopeptidase (MAP), it may be another amino acid. ２In this case, it is also a preferred embodiment of the present invention that the hydrophobic tag does not have any other reactive sites. In this embodiment, gelation of the block copolymer may be suppressed.

[0049] The molecular weight of the protein structure may be, for example, 1 kDa or more, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more. The molecular weight of the first segment and the molecular weight of the protein structure contained in the first segment may be 400 kDa or less, 360 kDa or less, 300 kDa or less, 250 kDa, 200 kDa, 150 kDa, 100 kDa, 70 kDa, 50 kDa, or 30 kDa or less.

[0050] The molecular weights referred to herein are values ​​measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Such electrophoresis is performed as follows: First, 200 μL of 2 M lithium chloride DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.) is added to 2 mg of powder sample, and the sample is dissolved by stirring at 80°C for 60 minutes and then at 95°C for 10 minutes. The sample is then diluted 50-fold with 10 M (mol / L) urea solution, further diluted 2-fold with sample buffer (Fujifilm Wako Pure Chemical Industries, Ltd.), and heated at 95°C for 5 minutes to denature the protein. Next, an SDS-PAGE gel (Bio-Rad) is attached to an electrophoresis apparatus (Bio-Rad), the apparatus is filled with SDS buffer, and the electrophoresis apparatus is connected to a power supply (Biocraft). Ten microliters of the denatured sample was added to each well of an SDS-PAGE gel, and a current of 30 mA was applied for 30 minutes. After electrophoresis, the SDS-PAGE gel was removed from the apparatus, immersed in Oriole fluorescent gel stain (Bio-Rad), and shaken for 1 hour. The gel was then placed on a UV sample tray (Bio-Rad), and a stained image was captured using a Gel Doc EZ gel imager (Bio-Rad).

[0051] The number of amino acid residues constituting the protein structure may be 50 or more. The number of amino acid residues may be, for example, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more. The number of amino acid residues may be, for example, 5000 or less, 4500 or less, 4000 or less, 3500 or less, 3000 or less, 2500 or less, 2000 or less, 1500 or less, or 1000 or less. The fewer the number of amino acid residues, the higher the solubility in a solvent tends to be. Therefore, when the number of amino acid residues of the protein structure according to this embodiment is, for example, 5000 or less or 2500 or less, when a compound containing a protein structure is dissolved in a solvent and reacted with a compound containing a molecular group having a plasticizing function for the protein structure to obtain a polymer compound containing a protein structure, it may be desirable to improve the efficiency of producing a polymer compound containing a target protein structure. In members such as films made using the polymer compound containing a protein structure obtained in this way, it can be expected that the flexibility will be improved while a certain degree of strength is ensured.

[0052] The protein structure may be a recombinant protein structure or a hydrophobic protein structure, which may be a hydrophobic recombinant protein structure.

[0053] A recombinant protein structure refers to a protein structure produced using genetic recombination technology. When the protein structure is a recombinant protein structure, the amino acid sequence can be easily modified, making it easier to control the characteristics and physical properties of polymer compounds containing the protein structure. When the protein structure is a recombinant protein structure, uniform molecular design is always possible, making it possible to stably obtain protein structures tailored to the intended purpose. This advantageously stabilizes the quality of polymer compounds and components such as films containing the desired protein structure.

[0054] When the protein structure is a hydrophobic protein structure, the affinity of the molecular group having a plasticizing function with the first segment is improved, making it possible to produce a member such as a film with greater flexibility. In addition, the water resistance of such a member is improved, which can advantageously extend the service life of the member, for example, when used as a general-purpose industrial material. The hydrophobicity or hydrophilicity of the entire polymer compound containing a protein structure can be adjusted arbitrarily, for example, by controlling the hydrophobicity or hydrophilicity of the molecular group having a plasticizing function for the protein structure, which is contained in the second segment. When the protein structure is a hydrophobic protein structure, the entire polymer compound containing the protein structure can be shifted toward the hydrophobic side compared to when the protein structure is a hydrophilic protein structure, thereby making it possible to control the hydrophobicity or hydrophilicity of the entire polymer compound containing the protein structure over a wider range.

[0055] The hydrophobicity of a hydrophobic protein structure can be estimated using the value of the average hydropathy index (degree of hydrophobicity: hydrophobicity index) described below as an index. The value of the average hydropathy index of a hydrophobic protein structure may be, for example, 0.00 or more, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more. The upper limit is not particularly limited, but may be, for example, 1.00 or less or 0.70 or less.

[0056] The hydrophobic protein structure preferably has low solubility in an aqueous lithium bromide solution (concentration: 9 M) at 60°C. This solubility can be evaluated using a protein structure obtained by decomposing a compound (protein structure) corresponding to the hydrophobic protein structure or a polymer compound containing the protein structure and isolating only the hydrophobic protein structure. The maximum concentration of the protein structure when dissolved in an aqueous lithium bromide solution (concentration: 9 M) at 60°C may be, for example, less than 30% by mass, less than 25% by mass, less than 20% by mass, less than 15% by mass, less than 10% by mass, less than 5% by mass, or less than 1% by mass. The hydrophobic protein structure may also be completely insoluble in an aqueous lithium bromide solution (concentration: 9 M) at 60°C.

[0057] The hydrophobic protein structure preferably has a large water contact angle. The water contact angle can be evaluated by forming a film on a substrate, the film being composed of a protein structure obtained by decomposing a compound (protein structure) corresponding to the hydrophobic protein structure or a polymer compound containing the protein structure and isolating only the hydrophobic protein structure, and using the film. A protein structure that constitutes a film that has a contact angle of 55° or more after 5 seconds of water being dropped onto the film is preferred as the hydrophobic protein structure. The contact angle may be, for example, 60° or more, 65° or more, or 70° or more.

[0058] The hydrophobic protein structure preferably has excellent hot water resistance. Hot water resistance can be evaluated using a protein structure obtained by decomposing a compound (protein structure) corresponding to the hydrophobic protein structure or a polymer compound containing the protein structure and isolating only the hydrophobic protein structure. A preferred hydrophobic protein structure is a protein that does not decompose even when a dispersion containing the protein structure and water is prepared, the dispersion containing the protein structure at 5% by mass, and is heated at 100°C for 5 hours.

[0059] <Second Segment> The second segment includes a molecular group having a plasticizing function for the protein structure. A molecular group having a plasticizing function for the protein structure refers to a molecular group in which the intermolecular force between the molecular groups is weaker than the intermolecular force between the protein structures, and when the two are mixed, the flexibility of the material can be improved compared to the protein structure alone. A molecular group having a plasticizing function for the protein structure can also be said to be a molecular group having a melting point or glass transition temperature lower than that of the protein structure. The molecular group having a plasticizing function for the protein structure may be, for example, polyether, polyester, or polycarbonate. The polyether as the molecular group may be, for example, polyethylene glycol (PEG) or polytetramethylene glycol (PTMG). The plasticizing function can also be said to be a function of improving flexibility or a function of increasing breaking elongation in bending and / or tension. A biodegradable molecular group or a biomass-derived molecular group is preferably used as the molecular group having a plasticizing function for the protein structure. This is expected to further increase biodegradability and biovalue, and further reduce the energy required for production.

[0060] The second segment may contain a plurality of the molecular groups. The second segment may contain a plurality of the molecular groups, and the plurality of molecular groups may be connected to each other. The connections between the molecular groups may be partially branched. By partially branching the connections, the second segment can have a branched structure consisting of a plurality of the molecular groups, and each branch can form multiple bonds with the first segment. In other words, by introducing branches into some of the connections, a network structure between the first segment and the second segment can be formed. In this way, when the second segment contains a plurality of the molecular groups and these molecular groups are connected to each other, the design options for the second segment can be further expanded, and the flexibility of a member such as a film can be more easily adjusted, for example. When the second segment contains only one of the molecular groups, multiple second segments may be connected to each other, and at least one of the multiple second segments may be bonded to the first segment. Alternatively, multiple second segments may be bonded to one first segment. When connecting a plurality of second segments together, a branch may be introduced into a portion of the connection between the plurality of second segments to form a network of second segments.

[0061] The second segment may include, for example, a skeleton derived from at least one selected from the group consisting of polyether, polyester, polycarbonate, polyamide, polyol (such as polyvinyl alcohol), polyolefin, polyacetal, polyketal, poly(meth)acrylate, silicone, polyurethane, polyalkyleneimine, phenolic resin, urea resin, melamine resin, and polysaccharide. Preferably, the second segment may include at least one functional group selected from the group consisting of a polyether group, a polyester group, a polycarbonate group, a polyamide group, a polyol group (such as a polyvinyl alcohol group), and a modified polysaccharide group, or may include at least one functional group selected from the group consisting of an ether group, an ester group, a carbonate group, an amide group, and a modified polysaccharide group. The second segment may include, for example, at least one structural unit selected from the group consisting of structural units having an ether bond, an ester bond, a carbonate ester bond (carbonate bond), an amide group, a siloxane bond, a urethane bond, a urethane bond, or a urea bond, and can also be said to include at least one structural unit selected from the group consisting of structural units having an alkylene group, a substituted alkylene group, an oxymethylene group, an alkyleneimine group, or a modified polysaccharide group.

[0062] Examples of polyether groups include functional groups derived from polyalkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene oxide / propylene oxide copolymer, and polybutylene glycol (polytetramethylene glycol (PTMG)). When these polyether groups are contained in the second segment, the polyether group may be directly bonded to a heteroatom (O, N, S) in an ester group, thioester group, or amide group of a linker (described below) contained in the second segment as needed. Alternatively, the polyether group may be directly bonded to a heteroatom (O, N, S) in an ester group, thioester group, or amide group of a protein structure contained in the first segment. In this case, the entire polyether group is more likely to separate from the linker and / or the first segment, thereby increasing the biodegradation rate of the polyether group.

[0063] Examples of the polyester group include functional groups derived from polyesters such as polylactic acid, poly(3-hydroxybutanoic acid), polyhydroxybutanoic acid / hydroxyvaleric acid copolymer, polyhydroxybutanoic acid / 4-hydroxybutanoic acid copolymer, polyhydroxybutanoic acid / hydroxyhexanoic acid copolymer, polytrimethylene terephthalate, butanediol / long-chain dicarboxylic acid copolymer, polyethylene terephthalate, polybutylene succinate, polybutylene succinate-adipate copolymer, polybutylene adipate-terephthalate copolymer, polycaprolactone, and polytrimethylene furandicarboxylate (PTF). Of the above polyesters, the polyester group is preferably a functional group derived from a material classified as a biomass plastic or biodegradable plastic, such as polycaprolactone.

[0064] Examples of the polycarbonate group include functional groups derived from polycarbonates having an aliphatic hydrocarbon chain as the main skeleton, such as 1,6-hexanediol polycarbonate, 1,5-pentanediol polycarbonate, and 1,10-decanediol carbonate.

[0065] Examples of the polyamide group include functional groups derived from polyamides such as nylon 3, nylon 4, nylon 5, nylon 6, nylon 11, and nylon 610. Of the above-mentioned polyamides, the polyamide group is preferably a functional group derived from a polyamide classified as a biomass plastic or a biodegradable plastic.

[0066] Examples of the polyol group (such as a polyvinyl alcohol group) include functional groups derived from polyols (such as polyvinyl alcohols) such as polyvinyl alcohol and ethylene-vinyl alcohol copolymers. Of the above-mentioned polyols, the polyol group is preferably a functional group derived from a polyol classified as a biomass plastic or a biodegradable plastic.

[0067] The modified polysaccharide group may be, for example, a functional group derived from a compound obtained by chemically modifying cellulose, starch, chitin, chitosan, etc. Examples of the chemically modified compound include cellulose acetate, ethyl cellulose, starch acetate, hydroxypropylated starch, carboxymethyl chitin, and carboxymethyl chitosan.

[0068] The second segment may further include a linker in addition to the molecular group having a plasticizing function for the protein structure. In this case, the molecular group and the protein structure may be bonded via the linker. Examples of the linker include groups formed by the reaction of electrophilic functional groups represented by the formulas (1) to (20) described below with the above-mentioned reactive sites.

[0069] The reactive site in the first segment is a segment that reacts with the second segment and a compound having a reactive group. The reactive group is preferably an electrophilic functional group represented by any of formulas (1) to (20) described below. Among these, it is preferable that the reactive site is a thiol group and the reactive group is a thiol-reactive group. The thiol-reactive group is preferably a group having an ethylenically unsaturated bond, and more preferably a group represented by formula (M-1) or formula (M-2) described below.

[0070] The molecular weight of the second segment (a molecular group having a plasticizing function on the protein structure) may be, for example, 200 to 500,000, 300 to 400,000, 350 to 350,000, 400 to 300,000, 500 to 200,000, 600 to 100,000, 700 to 50,000, 800 to 10,000, 900 to 7,500, or 1,000 to 5,000.

[0071] When the molecular weight of the second segment is 200 or more, localization of the molecular group having a plasticizing function within the three-dimensional structure of the molecule is easily suppressed, and the mass ratio of the molecular group having a plasticizing function that must be introduced to exert a certain level of function can be sufficiently reduced. As a result, it may be possible to shorten the reaction time and lower the reaction temperature in the production of a polymer compound containing a protein structure.

[0072] When the molecular weight of the second segment is 500,000 or less, the molecular weight falls within an appropriate range, and a decrease in the binding reactivity of the second segment with the first segment is more easily suppressed.

[0073] The molecular weight of the second segment is a weight average molecular weight, which is generally determined by a known method using GPC.

[0074] The molecular weight of the second segment (a molecular group having a plasticizing function for the protein structure) relative to the molecular weight of the first segment (protein structure) can be adjusted appropriately depending on the application of the polymer compound containing the protein structure, etc. The molecular weight of the second segment (the total molecular weight when two or more second segments are bonded to one first segment) is, for example, preferably 1 to 10,000, more preferably 1.5 to 9,000, even more preferably 2 to 8,000, still more preferably 3 to 7,000, even more preferably 5 to 5,000, still more preferably 7 to 3,000, and even more preferably 10 to 2,000, based on the molecular weight of the first segment (100).

[0075] When the ratio of the molecular weight of the second segment to the molecular weight of the first segment is 1 or more, the flexibility of a member such as a film obtained using a polymer compound containing a protein structure can be further increased. When the ratio of the molecular weight of the second segment to the molecular weight of the first segment is 10,000 or less, the member has sufficient plasticity (flexibility) and can further improve the rigidity of the material of the member such as a film. When the ratio of the molecular weight of the second segment to the molecular weight of the first segment is within the above-mentioned range, it is possible to produce a member such as a film with excellent flexibility.

[0076] The molecular weight of the second segment and the molecular weight of the molecular group having a plasticizing function for the protein structure may be, for example, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more, 5.0 or more, 10 or more, 20 or more, 30 or more, or 40 or more, when the molecular weight of the first segment or the molecular weight of the protein structure contained in the first segment is taken as 100 (preferably when the molecular weight of the protein structure contained in the first segment is taken as 100). The upper limit is not particularly limited, and may be, for example, 1000 or less, 800 or less, 600 or less, 400 or less, 200 or less, 100 or less, 80 or less, 70 or less, 60 or less, or 50 or less. The molecular weight of the second segment, when the molecular weight of the first segment is taken as 100, is preferably in the range of 1 to 1,000, more preferably in the range of 1 to 800, even more preferably in the range of 1 to 600, even more preferably in the range of 1 to 400, even more preferably in the range of 1 to 200, even more preferably in the range of 1 to 100, preferably in the range of 1 to 70, more preferably in the range of 1.5 to 60, even more preferably in the range of 1.5 to 50, and particularly preferably in the range of 2.0 to 50. When the ratio of the molecular weight of the first segment (protein structure) to the molecular weight of the second segment (molecular group having a plasticizing function for the protein structure) is a value within the above range, for example, in a member such as a film obtained using a polymer compound containing a protein structure, it can be expected that the properties of the first segment due to the presence of the protein structure (e.g., high mechanical strength) will be sufficiently maintained while improving flexibility, extensibility, etc. The ratio of the molecular weight of the second segment when the molecular weight of the first segment is taken as 100 is determined in terms of weight average molecular weight.

[0077] The content ratio of the first segment (protein structure) to the second segment (molecular group having plasticizing function) in the polymer compound containing a protein structure can be adjusted appropriately depending on the application of the member such as a film. When the second segment is taken as 100, the content ratio of the first segment may be, for example, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, or 600 or more. The upper limit of this value is not particularly limited, but may be 1000 or less, 900 or less, 800 or less, or 700 or less. By setting the content ratio of the first segment to the second segment in the polymer compound containing a protein structure to a value within the above range, wasteful use of the second segment can be suppressed, thereby reducing the production cost of the polymer compound containing a protein structure. In a polymer compound containing a protein structure, the content ratio of the second segment to the first segment can be advantageously reduced by, for example, reducing the molecular weight of the second segment.

[0078] In the above-described embodiments, the linker has been described as a component of the second segment, but it may be treated separately from the second segment. The linker can also be treated as a component of the first segment. Although the molecular weight of the linker portion is smaller than that of the protein structure and the molecular group, when the linker is treated separately from the second segment, the range obtained by subtracting the molecular weight of the linker portion from the molecular weight of the second segment is treated as the preferred molecular weight range of the second segment. Similarly, when the linker portion is treated as a component of the first segment, the range obtained by adding the molecular weight of the linker portion to the molecular weight of the first segment is treated as the preferred molecular weight range of the first segment.

[0079] <Block Copolymer> The number of second segments in the block copolymer is one or more, preferably two or more, more preferably two to ten, even more preferably two to eight, particularly preferably two to six, and most preferably two to four, per one first segment.

[0080] The block copolymer having a first segment and a second segment may be, for example, a block copolymer in which one or more second segments are bonded to one first segment, or a block copolymer in which multiple blocks each including a first segment and a second segment bonded to the first segment are linked together. The block copolymer contained in the polymer compound containing a protein structure may be a polymer (e.g., a graft polymer) having a first segment containing a protein structure as the main chain and a second segment as a side chain. The block copolymer may, for example, have multiple first segments, in which the first segments and the second segments are bonded alternately. The alternate bonding of the first segments and the second segments can further improve the elongation and toughness of components such as fibers and films.

[0081] The block copolymer may have a plurality of the first segments, and a portion of the second segment may be bonded to two or more of the first segments to form a network structure, which can further improve the elongation and toughness of members such as fibers and films.

[0082] The bond between the first segment and the second segment may be a direct bond, or may be a bond via a structure that allows bonding between the first segment and the second segment. In any of these bonding forms, the bond between the first segment and the second segment may be a coordinate bond, a bond due to ionic interaction, or a covalent bond. Among these, a covalent bond is preferable.

[0083] [Method for Producing Block Copolymer] The method for producing a block copolymer according to this embodiment is a method for producing a block copolymer by combining a compound having a first segment including a protein structure with a compound having a second segment containing a molecular group having a plasticizing function for the protein structure, wherein the protein structure includes a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and has at least two reactive sites that react with the second segment and a compound having a reactive group, and the most N-terminal reactive site among the reactive sites is present within the hydrophobic tag on the N-terminus side or is present at the N-terminus of the protein structure, NH ２ or is present within the main sequence, and the reactive site closest to the C-terminus of the reactive sites is present within the hydrophobic tag on the C-terminus side, or is a protein structure present within 9 residues from the C-terminus of the main sequence.

[0084] Preferred aspects of the first segment and the second segment in the method for producing a block copolymer according to this embodiment are the same as the preferred aspects of the first segment and the second segment in the block copolymer according to this embodiment described above. Furthermore, preferred aspects of the block copolymer produced by the method for producing a block copolymer according to this embodiment are the same as the preferred aspects of the block copolymer according to this embodiment described above.

[0085] In the method for producing a block copolymer according to this embodiment, as described above, the reactivity of the compound having a first segment containing a protein structure is high, and therefore the bonding between the compound having a first segment containing a protein structure and the compound having a second segment containing a molecular group capable of plasticizing the protein structure proceeds quickly (the reaction rate is high), resulting in a method with excellent productivity for block copolymers.

[0086] Furthermore, the method for producing a block copolymer according to this embodiment includes adding a compound having the first segment and a compound having the second segment to a solvent, and the compound having the second segment is preferably added in a non-solution state. The compound having the second segment is preferably a compound having a second segment and a reactive group. Preferred aspects of such compounds are as described above. The non-solution state refers to a state in which the compound is not dissolved in a solvent, and is preferably a solid or liquid state. Another preferred aspect of the present invention is one in which, in the non-solution state, the compound having the second segment is a single compound rather than a mixture with other additives, etc. According to the above aspect, a block copolymer can be produced by adding the compound having the first segment and the compound having the second segment to one reaction vessel.

[0087] The compound having the first segment according to this embodiment is highly reactive, and the amount of the compound having the first segment reacting with itself is small, so that the reaction can proceed quickly even when the compound having the second segment is added in a non-solution state.

[0088] In the above-mentioned aspect, one preferred aspect also includes adding a reducing agent, a compound having the first segment, and a compound having the second segment to the solvent in this order.

[0089] -Reducing Agent- The reducing agent may include, for example, at least one selected from the group consisting of thiol, dithiol, sodium carbonate, sodium sulfite, sodium hyposulfite, sodium sulfate, and sodium dithionite. The reducing agent may include at least one selected from the group consisting of dithiol, sodium carbonate, and sodium sulfite, and may be dithiol or sodium sulfite. As the reducing agent, a compound having a mercapto group is preferred.

[0090] The compound having a mercapto group is preferably a compound represented by the following general formula (Y). Such a compound forms a chemically stable six-membered ring structure in its oxidized form after the reduction reaction, suppressing the reaction between the reducing agent and the reactive group in the compound having a second segment and a reactive group in the reaction system. Furthermore, by forming a ring structure between the reducing agent and a reactive site on the protein, for example, the formation of bonds such as disulfide bonds between proteins is suppressed, making the reaction between the mercapto group on the protein and the reactive group more significant, allowing for more efficient production of the target polymer compound in accordance with the reaction design. Examples of dithiols include dithiothreitol and 1,4-butanedithiol. The compound (dithiol) represented by general formula (Y) is specifically dithiothreitol or 1,4-butanedithiol.

[0091]

[0092] Other compounds having a mercapto group include, for example, 3-mercaptopropionic acid, 3-mercapto-1,2-propanediol, and pentaerythritol tetra(3-mercaptopropionate). In addition to the above compounds, the reducing agent may also be, for example, tris(2-carboxyethyl)phosphine (TCEP).

[0093] The lower limit of the amount of the reducing agent may be, for example, 0.5 equivalents or more, 0.8 equivalents or more, or 1.0 equivalents or more relative to the reactive sites of the compound having the first segment. By setting the lower limit of the amount of the reducing agent within the above range, the formation of bonds such as disulfide bonds due to reactions between reactive sites can be more sufficiently inhibited. This can further improve the production efficiency of the target synthetic polymer compound. The upper limit of the amount of the reducing agent may be, for example, 2.4 equivalents or less, 2.0 equivalents or less, 1.6 equivalents or less, 1.2 equivalents or less, or 1.1 equivalents or less relative to the reactive sites. By setting the upper limit of the amount of the reducing agent within the above range, the reaction can proceed while further suppressing the reaction between the reactive group and the reducing agent and the generation of by-products.

[0094] In the method for producing a block copolymer of the present invention, other additives may be present when bonding a compound having a first segment containing a protein structure with a compound having a second segment containing a molecular group capable of plasticizing the protein structure. Examples of other additives include a base and a polymerization inhibitor.

[0095] <<Base>> By including a base, the reactive site can be activated and the reaction with the reactive group can be promoted. Examples of the base include primary amines, secondary amines, tertiary amines, nitrogen-containing ring compounds, nitrogen-containing aromatic compounds, and inorganic bases. Examples of primary amines include ethanolamine and hexamethylenediamine. Examples of secondary amines include diethylamine, methylethylamine, and N-methylbutylamine. Examples of tertiary amines include triethylamine, diethylmethylamine, and DIPEA (N,N-diisopropylethylamine). Examples of nitrogen-containing ring compounds include quinuclidine, DABCO (1,4-diazabicyclo[2.2.2]octane), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), and DBN (1,5-diazabicyclo[4.3.0]non-5-ene). Examples of nitrogen-containing aromatic compounds include pyridine, imidazole, etc. Examples of inorganic bases include sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, cesium carbonate, tripotassium phosphate, sodium acetate, potassium acetate, etc.

[0096] The lower limit of the amount of the base may be, for example, 0.01 parts by mass or more, 0.05 parts by mass or more, 0.1 parts by mass or more, or 0.5 parts by mass or more, based on the mass of the compound having the first segment. By setting the lower limit of the amount of the base within the above range, the reaction can be further promoted. The upper limit of the amount of the base may be, for example, 50 parts by mass or less, 20 parts by mass or less, 10 parts by mass or less, or 5 parts by mass or less, based on the mass of the compound having the first segment. By setting the upper limit of the amount of the base within the above range, molecular decomposition due to the influence of residual base can be further suppressed, allowing the synthesis of the desired polymer compound. The amount of the base may be adjusted within the above range and may be, for example, 0.05 to 50 parts by mass or 0.1 to 10 parts by mass, based on the mass of the compound having the first segment.

[0097] <<Polymerization Inhibitor>> The polymerization inhibitor is a compound that inhibits self-polymerization of the compound having the second segment and the reactive group. Examples of the polymerization inhibitor include hydroquinone, methoquinone, 4-tert-butylpyrocatechol, tert-butylhydroquinone, 1,4-benzoquinone, dibutylhydroxytoluene, methoquinol, phenothiazine, and 1,1-diphenyl-2-picrylhydrazyl.

[0098] The content of the polymerization inhibitor may be, for example, 0.01 parts by mass or more, 0.05 parts by mass or more, 0.1 parts by mass or more, or 0.2 parts by mass or more, relative to 100 parts by mass of the compound having a second segment and a reactive group. The content of the polymerization inhibitor may be, for example, 25 parts by mass or less, 10 parts by mass or less, 5 parts by mass or less, or 1 part by mass or less, relative to 100 parts by mass of the compound having a second segment and a reactive group. By setting the content of the polymerization inhibitor within the above range, it is possible to further suppress the polymerization inhibitor from inhibiting the reaction in the system.

[0099] Furthermore, since the amount of the compound having the first segment reacting with itself is small, it is possible to reduce the amount of the reducing agent. That is, the method for producing a block copolymer according to this embodiment can be a simple process using only one reaction vessel. Conventional methods for producing block copolymers involve adding a solution of a compound having a second segment dissolved in a solvent to a solution of a compound having a first segment dissolved in a solvent. This method requires two vessels: one for dissolving the compound having the first segment in the solvent, and another for dissolving the compound having the second segment in the solvent. Considering an actual mass production process, reducing the number of vessels used for reaction or preparation can be an industrially very important improvement, as it facilitates the design of processes and equipment.

[0100] -First Aspect of Method for Producing Block Copolymer- The block copolymer is preferably obtained by a production method including a step of mechanochemically treating a mixture containing a compound having a first segment containing a protein structure (hereinafter also referred to as "compound A") and a compound having a second segment containing a molecular group capable of plasticizing the protein structure (hereinafter also referred to as "compound B"). The mixture may be prepared by mixing a solution containing compound A and a solvent with a solution containing compound B and a solvent, or, as described above, may be prepared by sequentially adding compound A and compound B in a non-solution state to a solvent. The mixture may further contain the above-mentioned reducing agent, base, polymerization inhibitor, etc. The first segment is formed by compound A, and the second segment is formed by compound B. That is, the block copolymer is preferably obtained by mechanochemically treating a mixture containing the compound having the protein structure and a compound having a molecular group capable of plasticizing the protein structure.

[0101] The preferred embodiment of the protein structure in Compound A is as described above.

[0102] Compound B is preferably a compound having a second segment and a reactive group. Preferred embodiments of such a compound are as described above.

[0103] The reactive group in the compound having the second segment and the reactive group is, for example, a group represented by formula (1) to (20), and a preferred reactive group is a group represented by formula (1), (2), (6) to (8), (16), or (19), and a more preferred reactive group is a group represented by formula (1), (2), or (6). Preferred combinations of reactive sites (reactive sites contained in the protein structure of compound A) and reactive groups are a hydroxyl group and a hydroxy-reactive group represented by any of formulas (2) to (9), (13) to (17), and (19), an amino group and an amine-reactive group represented by any of formulas (2) to (9), (13) to (17), and (19), and a thiol group and a thiol-reactive group represented by any of formulas (1) to (6), (8), (10) to (15), and (18).

[0104] In formulas (1) to (20), R ２ In formulae (2) to (8) and (13) to (15), Y represents, independently of each other, an oxygen atom, a sulfur atom, or NR １ indicates R １ represents a hydrogen atom, a hydrocarbon group, an aromatic group, a carbonyl group, or a sulfonyl group. In formulas (2) to (6) and (10) to (15), R each independently represents a hydrogen atom, a hydrocarbon group, or an aromatic group. In formulas (8) and (16), Z represents a halogen atom, a sulfonate ester group, or a fluorine-containing carboxylate ester group. In formula (19), X represents a halogen atom.

[0105] The amount of compound B used may be 1.5 to 7 equivalents per nucleophilic functional group contained in compound A. The amount of compound B used may preferably be 1.5 to 6.5 equivalents, 1.5 to 6 equivalents, 1.5 to 5.5 equivalents, 1.5 to 5 equivalents, 1.5 to 4 equivalents, 2 to 7 equivalents, 2 to 6.5 equivalents, 2 to 6 equivalents, 2 to 5.5 equivalents, 2 to 5 equivalents, 2 to 4.5 equivalents, 2.5 to 7 equivalents, 2.5 to 6.5 equivalents, 2.5 to 6 equivalents, 2.5 to 5.5 equivalents, 2.5 to 5 equivalents, or 2.5 to 4.5 equivalents per nucleophilic functional group. "Equivalent per nucleophilic functional group" means the molar equivalent of compound B per nucleophilic functional group contained in compound A. By setting the amount of compound B used to a value within the above range, the reactivity or reaction efficiency between compound A and compound B in the mechanochemical treatment is enhanced, and the target block copolymer can be obtained more efficiently.

[0106] Mechanochemical processing is a process in which a chemical reaction is induced by the direct absorption of mechanical energy. Such mechanical energy may be exerted, for example, by impact force or shear force. Specifically, such mechanochemical processing is carried out using a tumbling ball mill, a media-agitated mill, a planetary mill, a jet mill, a mixer mill, an extruder (twin-screw extruder), or the like. Specifically, compound A and compound B are placed in a grinding jar, and media balls, etc., are added depending on the type of mill used. The grinding jar is then placed in a mixer mill device and vibrated at a predetermined frequency and reaction time. At this time, a solvent, a base, a reaction accelerator, etc. may be further added to the grinding jar. Alternatively, compound A and compound B are placed in a mixer mill, and the milling procedure is initiated, followed by continuous kneading of the reactants. At this time, a solvent, a base, a reaction accelerator, etc. may be further added to the mixer mill. Alternatively, compound A and compound B are placed in an extruder and kneaded. At this time, a solvent, a base, a reaction accelerator, etc. may be further added to the extruder. Furthermore, if mechanochemical treatment is carried out using an extruder, it becomes possible to continuously produce the desired block copolymer.

[0107] The frequency can be adjusted appropriately by those skilled in the art depending on the reaction, and may be, for example, 10 to 50 Hz, 10 to 45 Hz, 10 to 40 Hz, 10 to 35 Hz, 15 to 50 Hz, 15 to 45 Hz, 15 to 40 Hz, 15 to 35 Hz, 20 to 50 Hz, 20 to 45 Hz, 20 to 40 Hz, or 20 to 35 Hz.

[0108] The reaction time can be set to the point where the raw material compound A disappears or a certain level of the block copolymer is detected by infrared (IR) absorption spectroscopy, gel filtration chromatography (GPC), etc. The reaction time may be any time, for example, 30 to 240 minutes, 30 to 210 minutes, 30 to 180 minutes, 30 to 150 minutes, 30 to 120 minutes, 30 to 110 minutes, 30 to 100 minutes, 30 to 95 minutes, 60 to 240 minutes, 60 to 210 minutes, 60 to 180 minutes, 60 to 150 minutes, 60 to 120 minutes, 60 to 110 minutes, 60 to 100 minutes, 6 ... The time may be up to 95 minutes, 70 to 240 minutes, 70 to 210 minutes, 70 to 180 minutes, 70 to 150 minutes, minutes, 70 to 120 minutes, 70 to 110 minutes, 70 to 100 minutes, 70 to 95 minutes, 80 to 240 minutes, 80 to 210 minutes, 80 to 180 minutes, 80 to 150 minutes, minutes, 80 to 120 minutes, 80 to 110 minutes, 80 to 100 minutes, or 80 to 95 minutes.

[0109] The solvent may be any solvent capable of swelling compound A or capable of dissolving at least one of compound A and compound B. However, it must be a compound that is liquid at room temperature and normal pressure and does not chemically react with compound A. Mechanochemical treatment using such a solvent is known as liquid-assisted grinding (LAG), and is a method in which the desired reaction proceeds more efficiently by adding a small amount of solvent to a grinding jar. Examples of such solvents include alcoholic solvents such as methanol and ethanol, and aprotic polar solvents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpiperidone (NMP), and dihydrolevoglucosenone. The amount of solvent used may be, for example, 0.01 g to 1 g, or may be 0.01 g to 0.8 g, 0.01 g to 0.6 g, 0.01 g to 0.4 g, or 0.01 g to 0.2 g per 1 g of compound A. In other words, the amount of solvent used may be, for example, 1 to 100% by mass, or may be 1 to 80% by mass, 1 to 60% by mass, 1 to 40% by mass, or 1 to 20% by mass, relative to compound A. It is believed that a trace amount of solvent forms a microscopic reaction field by swelling compound A or locally dissolving compound A. Note that the addition of a solvent is not always necessary; for example, when compound B is a liquid at room temperature and normal pressure and has the above-mentioned function as a solvent, the addition of a solvent may not be necessary.

[0110] After the reaction is complete, the mixture may be removed from the grinding jar and washed with a solvent. Washing can remove unreacted substances and excess by-products produced by the reaction. Examples of solvents used for washing include water, methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, ethyl acetate, and hexane. After washing, the product may be dried to distill off the solvent used for washing. Drying may be performed under reduced pressure.

[0111] The average particle size of the block copolymer according to this embodiment is preferably 1 to 80 μm, 1 to 50 μm, 2 to 50 μm, 2 to 40 μm, 4 to 25 μm, 5 to 25 μm, 8 to 25 μm, or 8 to 16 μm. An average particle size within the above range provides excellent handleability. The average particle size can be determined, for example, by the following method. Particles or powder of the block copolymer are uniformly dispersed on a glass plate by suction in a vacuum chamber, and then measured five times each using a wet / dry image analysis particle size distribution meter (product name: DW-200 nano, manufactured by Jasco International Inc.). Projected images are then captured using a 10-megapixel camera, and the resulting projected images are analyzed using image analysis software.

[0112] The block copolymer according to this embodiment is obtained as a powder with a smaller average particle size and a more uniform particle size by mechanochemically treating a mixture containing compound A and compound B and then freeze-drying it. The average particle size of the block copolymer powder obtained by freeze-drying is, for example, about 1 to 30 μm, 1 to 20 μm, 1 to 10 μm, or 2 to 8 μm. Such a powdered block copolymer is easier to handle and allows for fine adjustment of the amount used. Moreover, the freeze-dried block copolymer not only has a small average particle size, but also is expected to loosen the tertiary structure of compound A, thereby improving its dispersibility in aqueous media (aqueous liquids) such as water, basic aqueous solutions, acidic aqueous solutions, and neutral aqueous solutions containing inorganic salts, as well as its solubility in solvents. In particular, when the block copolymer powder obtained by mechanochemical treatment or an aqueous dispersion obtained by dispersing the block copolymer powder obtained by further freeze-drying it in an aqueous medium is applied to a substrate surface, it can be applied more uniformly and without unevenness. This allows for the formation of a more uniform coating film with as little unevenness as possible. That is, the aqueous dispersion of the block copolymer powder can also be useful as a coating liquid for forming a coating film. Furthermore, the aqueous dispersion of the block copolymer powder can be applied to the surface of a substrate, followed by laminating another substrate thereon, and then curing the block copolymer to bond the two substrates. In other words, the aqueous dispersion of the block copolymer according to this embodiment can also be useful as a water-dispersible adhesive. When the aqueous dispersion of the block copolymer is used as a coating liquid, for example, a predetermined colorant or an additive commonly used in coating liquids or the like may be added, blended, or mixed with the aqueous dispersion of the block copolymer. When the aqueous dispersion of the block copolymer is used as a water-dispersible adhesive, an additive commonly used in adhesives or the like may be added, blended, or mixed with the aqueous dispersion of the block copolymer. Since the coating liquid and water-dispersible adhesive made from the aqueous dispersion of the block copolymer according to this embodiment use an aqueous medium such as water as a solvent, they are suitable for application to or adhesion of materials with low organic solvent resistance, and the odor characteristic of organic solvents can also be reduced.

[0113] Block copolymers produced by mechanochemical methods use no organic solvents or only a very small amount of organic solvent (an amount sufficient to swell molecules capable of plasticizing compound A or compound B) in the production process, so that organic solvents are less likely to remain and the copolymers can be easily purified into powders.

[0114] - Second Aspect of Method for Producing Block Copolymer - Furthermore, the block copolymer in the polymer compound containing a protein structure is also preferably produced by a method comprising heating and reacting a mixture containing: a compound having the above-mentioned first segment, wherein the above-mentioned reactive site is a mercapto group (hereinafter also referred to as "compound C"); a compound having at least one structure selected from the group consisting of polyethers, polyesters, and polycarbonates, which has two structures represented by the following formula (M-1) or formula (M-2) (hereinafter also referred to as "compound D"); and an organic solvent. In formula (M-1), M is H, Na, K, or NHEt ３ , or NHEtiPr ２ In this specification, Et represents an ethyl group, and iPr represents an isopropyl group. １ represents a hydrogen atom or a methyl group.

[0115] The mixture may be prepared by mixing a solution containing compound C and a solvent with a solution containing compound D and a solvent, or by sequentially adding compound C and compound D in a non-solution state to a solvent, as described above. The mixture may further contain the above-mentioned reducing agent, base, polymerization inhibitor, etc. The above-mentioned first segment is formed from compound C, and the above-mentioned second segment is formed from compound D.

[0116] <<Compound C>> Compound C is a compound containing the above-described first segment. Here, the mercapto group in Compound C is preferably a mercapto group contained in a cysteine ​​residue in a protein structure. Other preferred aspects of the first segment in Compound C are as described above.

[0117] In the above production method, the blending amount of the compound C may be, for example, more than 1 part by mass, 2 parts by mass or more, 5 parts by mass or more, more than 5 parts by mass, 6 parts by mass or more, 7 parts by mass or more, or 8 parts by mass or more, relative to 100 parts by mass of the organic solvent. In the production method according to the present disclosure, the blending amount of the compound C may be, for example, 50 parts by mass or less, 30 parts by mass or less, 20 parts by mass or less, or 15 parts by mass or less, relative to 100 parts by mass of dimethyl sulfoxide. When the upper limit of the blending amount of the compound C is within the above range, the reaction can proceed while more sufficiently suppressing the generation of by-products and gelation.

[0118] <<Compound D>> Preferred embodiments of the polyether, polyester, and polycarbonate in Compound D are the same as those preferred embodiments in the second segment described above.

[0119] In the above production method, the ratio of the number of moles of the structure represented by formula (M-1) or formula (M-2) in compound D to the number of moles of mercapto groups in compound C may be, for example, 0.1 times or more, 0.2 times or more, 0.5 times or more, or 0.7 times or more. By setting this ratio within the above range, the reaction can proceed while further suppressing a decrease in reactivity. Furthermore, in the above production method, the ratio of the number of moles may be, for example, 5.0 times or less, 3.0 times or less, 1.5 times or less, or 0.8 times or less. By setting this ratio within the above range, the reaction can proceed while further suppressing the generation of by-products and gelation. By adjusting the above molar ratio, the number of polyether structures, etc. in the obtained polymer compound can be adjusted.

[0120] <<Organic Solvent>> The organic solvent is not particularly limited as long as it can dissolve Compound C and Compound D, but dimethyl sulfoxide is preferred.

[0121] Here, when a compound having a structure represented by the above formula (M-1) is used as compound D, it is preferable to add a reducing agent during the heating reaction, and it is preferable to add a reducing agent and a base during the heating reaction. Furthermore, when a compound having a structure represented by the above formula (M-2) is used as compound D, it is preferable to add a polymerization inhibitor and a reducing agent during the heating reaction, and it is preferable to add a polymerization inhibitor, a reducing agent, and a base during the heating reaction. Preferred aspects of the reducing agent, the base, and the polymerization inhibitor are as described above.

[0122] <<Reaction Conditions>> In the above production method, the reaction temperature in the heating reaction may be, for example, 50°C or higher, 53°C or higher, 55°C or higher, 57°C or higher, or 60°C or higher. When the lower limit of the reaction temperature is within the above range, the reaction between compound C and compound D can be further promoted. In the above production method, the upper limit of the reaction temperature for the reaction between compound C and compound D may be, for example, 90°C or lower, 85°C or lower, 80°C or lower, 75°C or lower, or 70°C or lower. When the upper limit of the reaction temperature is within the above range, the formation of bonds between mercapto groups in proteins as a side reaction can be further suppressed, allowing the desired polymer compound to be synthesized efficiently. The reaction temperature for the reaction between compound C and compound D may be adjusted within the above range, and may be, for example, 50 to 90°C, 53 to 80°C, 55 to 75°C, or 55 to 70°C.

[0123] The timing of heating is not limited to after mixing of compounds C and D, and further components such as a base, a reducing agent, and a polymerization inhibitor that are added as needed. For example, these components may be added to a preheated organic solvent (e.g., dimethyl sulfoxide); necessary components out of the base, reducing agent, and polymerization inhibitor may be added to dimethyl sulfoxide, heating may be initiated, and after a predetermined temperature has been reached, compounds C and D may be added and reacted; or one of compounds C and D and necessary components out of the base, reducing agent, and polymerization inhibitor may be added, heating may be initiated, and after a predetermined temperature has been reached, the remaining one of compounds C and D may be added and reacted.

[0124] According to such a method, the block copolymer is obtained in the form of a solution dissolved in an organic solvent. The block copolymer is recovered from this solution by a known method such as drying, reprecipitation, or dialysis, and then pulverized as necessary to obtain a powder of a polymer compound containing a protein structure.

[0125] Alternatively, the block copolymer can be obtained by, for example, the method described in WO 2023 / 013638.

[0126] <Applications> The block copolymer of this embodiment can be used for synthetic leather, electronic materials, etc., and is preferably used as synthetic leather. As synthetic leather, it can be used for the same applications as conventional synthetic leather (for example, synthetic leather made of synthetic resin). The synthetic leather of this embodiment can be used for applications such as clothing, decorative items such as shoes and bags, various covers and furniture, and automotive interior materials. As electronic materials, it can be used for applications such as flexible flat cables, flexible printed wiring boards, insulating films, cover films, reinforcing plates, glass, and support members for silicon wafers, etc.

[0127] (Artificial Protein) The artificial protein of the present invention comprises a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and also comprises hydrophobic tags at the N-terminus and C-terminus, and has at least two cysteine ​​residues, the N-terminal most cysteine ​​residue of which is present in the main sequence, and the C-terminal most cysteine ​​residue of which is present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. Preferred embodiments of the artificial protein of the present invention are similar to the preferred embodiments of the protein structure in the block copolymer of the present invention described above, except that the reactive site is limited to the cysteine ​​residue (thiol group). By using the artificial protein of the present invention, the block copolymer of the present invention can be produced with high productivity. Furthermore, the artificial protein of the present invention comprises a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and also comprises hydrophobic tags at the N-terminus and C-terminus, and has at least one lysine residue, wherein the lysine residue closest to the N-terminus is present in the N-terminus hydrophobic tag, or there is no lysine residue in the N-terminus hydrophobic tag, and the lysine residue closest to the C-terminus is present in the C-terminus hydrophobic tag, or there is one lysine residue within 9 residues from the C-terminus of the main sequence. When there is no lysine residue in the N-terminus hydrophobic tag, the NH ２ can function as a reactive site. Preferred embodiments of the artificial protein of the present invention are the same as the preferred embodiments of the protein structure in the block copolymer of the present invention described above, except that the reactive site is limited to a lysine residue (amino group). By using the artificial protein of the present invention, the block copolymer of the present invention can be produced with good productivity. Furthermore, even when the reactive site is limited to a lysine residue, the artificial protein may contain a cysteine ​​residue that does not function as the reactive site.

[0128] The present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment details, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below. Hereinafter, M represents "mol / L", mM represents "mmol / L", and μM represents "μmol / L".

[0129] (Example 1: Examination of protein production yield and reactivity) <Production of protein (protein CTR) having a GFIL sequence at one end on the C-terminal side> [Preparation of polypeptide] (Preparation of expression vector) An artificial fibroin having the amino acid sequence (PRT2882) shown in SEQ ID NO: 9 was designed.

[0130] The average hydropathic index of the artificial fibroin having the amino acid sequence (PRT2882) shown in SEQ ID NO: 9 is 0.47.

[0131] Nucleic acids encoding artificial fibroins having the amino acid sequence shown in SEQ ID NO: 9 were synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. These nucleic acids were cloned into a cloning vector (pUC118). Subsequently, the nucleic acids were excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the polypeptide expression vector pET-22b(+) to obtain an expression vector.

[0132] (Preparation of Polypeptide) Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium containing ampicillin (Table 2) and measured at an absorbance (OD) of 600 nm. ６００ The culture temperature was kept at 30°C, and the OD ６００ The flask culture was continued until the fertilization rate reached 5 (about 15 hours), and a seed culture solution was obtained.

[0133]

[0134] The seed culture solution was added to a jar fermenter containing 500 mL of production medium (Table 3) and the OD ６００ The culture temperature was maintained at 37°C, and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration.

[0135]

[0136] Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, 120 g / 1 L of yeast extract) was added at a rate of 1 mL / min. The culture temperature was maintained at 37°C, and the culture was controlled to a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. 1 M isopropyl-β-thiogalactopyranoside (IPTG) was then added to the culture to a final concentration of 1 mM to induce expression of the artificial fibroin. 20 hours after the addition of IPTG, the culture was centrifuged, and the cells were collected. SDS-PAGE was performed using cells prepared from the cultures before and after the addition of IPTG. Expression of the desired artificial fibroin was confirmed by the appearance of a band of the desired artificial fibroin size, which was dependent on the addition of IPTG.

[0137] (Polypeptide purification) Bacterial cells harvested 20 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed bacterial cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF, and the cells were disrupted using a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until highly purified. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and dissolved by stirring with a stirrer at 60°C for 30 minutes. After dissolution, the solution was dialyzed against water using a dialysis tube (Cellulose tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.).

[0138] The white aggregated polypeptide obtained after dialysis was recovered by centrifugation, then dehydrated using a freeze-dryer. The freeze-dried powder was recovered to obtain powdered artificial fibroin (PRT2882). The formation of intermolecular disulfide bonds was confirmed for the artificial fibroin with cysteine ​​residues inserted. It was confirmed that the artificial fibroin contained 13.8 parts by mass of dimer, 2.1 parts by mass of trimer, and 0.8 parts by mass of tetramer per 100 parts by mass of monomer. Disulfide bond formation was measured by SDS-PAGE. This artificial fibroin (PRT2882) was used as the protein CTR.

[0139] <Production of a protein having GFIL sequences at both ends (protein A)> Powdered artificial fibroin (PRT3046) was obtained in the same manner as powdered artificial fibroin (PRT2882), except that the amino acid sequence (PRT3046) shown in SEQ ID NO: 10 was used instead of the amino acid sequence (PRT2882) shown in SEQ ID NO: 9. This artificial fibroin (PRT3046) was used as protein A.

[0140] <Production of Protein B> Powdered artificial fibroin (PRT3173) was obtained in the same manner as powdered artificial fibroin (PRT2882), except that the amino acid sequence (PRT3173) shown in SEQ ID NO: 11 was used instead of the amino acid sequence (PRT2882) shown in SEQ ID NO: 9. This artificial fibroin (PRT3173) was used as protein B.

[0141] <Production of Protein C> Powdered artificial fibroin (PRT3174) was obtained in the same manner as powdered artificial fibroin (PRT2882), except that the amino acid sequence (PRT3174) shown in SEQ ID NO: 12 was used instead of the amino acid sequence (PRT2882) shown in SEQ ID NO: 9. This artificial fibroin (PRT3174) was used as Protein C.

[0142] <Production of Protein D> Powdered artificial fibroin (PRT3175) was obtained in the same manner as powdered artificial fibroin (PRT2882), except that the amino acid sequence (PRT3175) shown in SEQ ID NO: 13 was used instead of the amino acid sequence (PRT2882) shown in SEQ ID NO: 9. This artificial fibroin (PRT3175) was used as Protein D.

[0143] <Production of Protein E> Powdered artificial fibroin (PRT3176) was obtained in the same manner as powdered artificial fibroin (PRT2882), except that the amino acid sequence (PRT3176) shown in SEQ ID NO: 14 was used instead of the amino acid sequence (PRT2882) shown in SEQ ID NO: 9. This artificial fibroin (PRT3176) was used as Protein E.

[0144] <Production of Protein F> Powdered artificial fibroin (PRT3177) was obtained in the same manner as powdered artificial fibroin (PRT2882), except that the amino acid sequence (PRT3177) shown in SEQ ID NO: 15 was used instead of the amino acid sequence (PRT2882) shown in SEQ ID NO: 9. This artificial fibroin (PRT3177) was used as Protein F.

[0145] <Production of Protein G> Powdered artificial fibroin (PRT3463) was obtained in the same manner as for powdered artificial fibroin (PRT2882), except that the amino acid sequence (PRT3463) shown in SEQ ID NO: 16 was used instead of the amino acid sequence (PRT2882) shown in SEQ ID NO: 9. This artificial fibroin (PRT3463) was used as Protein F.

[0146] Protein CTR is a protein in which the most N-terminal reactive point is located within the main sequence and the most C-terminal reactive point is located 10 residues from the C-terminus of the main sequence. Protein A is a protein in which the most N-terminal reactive point is located within the main sequence and the most C-terminal reactive point is located 10 residues from the C-terminus of the main sequence. Protein B is a protein in which the most N-terminal reactive point is not located within the main sequence and the most C-terminal reactive point is located 10 residues from the C-terminus of the main sequence. Protein C is a protein in which the most N-terminal reactive point is not located within the main sequence and the most C-terminal reactive point is located 3 residues from the C-terminus of the main sequence. Protein D is a protein in which the most N-terminal reactive point is not located within the main sequence and the most C-terminal reactive point is located at the C-terminus of the hydrophobic tag. Protein E is a protein in which the most N-terminal reactive point is located within the main sequence and the most C-terminal reactive point is located 3 residues from the C-terminus of the main sequence. Protein F is a protein in which the most N-terminal reactive site is present within the main sequence and the most C-terminal reactive site is present at the C-terminus of the hydrophobic tag. Protein G is a protein in which the most N-terminal reactive site is present at the N-terminus of the hydrophobic tag and the most C-terminal reactive site is present at the C-terminus of the hydrophobic tag.

[0147] [Method for measuring protein production amount] The purified product was freeze-dried to obtain a dry powder of the target protein. The weight of this powder was divided by the weight of the medium at the end of the culture to calculate the amount of powder obtained per 1 L of medium, and the production amount was compared. The measurement results are shown in the "Protein production amount" column in Figure 1. The larger the value, the higher the productivity of the artificial protein of the present invention.

[0148] [Method for measuring the amount of SS bonds] The amount of SS bonds was measured using gel filtration chromatography (GPC). Dried protein powder was analyzed in HFIP solution (final concentration: 1 mM) containing 0.1 wt% sodium trifluoroacetate. The sample was first dissolved using a heating and vibrating device (Front Lab MyBL-100S, AS ONE Corporation) at 45°C, 1500 rpm, for 1 hour, and then passed through a 0.45 μm pore size hydrophilic PTFE membrane filter (trade name: Dismic 25HP45AN, Advan Tech) to remove any remaining insoluble material. The GPC used was an Agilent 1260 Infinity II liquid chromatography system (Agilent Technologies, Inc.) equipped with a refractive index (RI) detector, a styrene-divinylbenzene copolymer column (inner diameter: 4.6 mm × 150 mm) equipped with a 0.5 μm guard column filter (trade name: Shodex GPC HK-G, manufactured by Showa Denko K.K.), and the mobile phase was monitored at a flow rate of 0.15 mL / min at 40°C using an Agilent 1260 Infinity II refractive index detector (RID) (Agilent Technologies, Inc.). A solution in which S—S bonds had been eliminated by reduction treatment was prepared as a control powder. The reduction treatment was carried out by adding TCEP (final concentration: 10 mM) to the sample solution before heating and shaking. For data analysis, the peak position of the reduced control sample was used as the reference, and the GPC peaks of each protein sample that had not been reduced were obtained. Peaks at the same position as the control sample's peak position were determined to represent monomers, and peaks appearing on the higher molecular weight side than the control sample's peak were determined to represent S-S bonds. The measurement results are shown in the "Amount of S-S bonds" column in Figure 1. The smaller the amount of S-S bonds, the higher the reactivity of the protein with the second segment.

[0149] [Production of Block Copolymer (BCP)] Proteins CTR and A to F (2 g, 202 μmol (Protein CTR), 186 μmol (Proteins A to F)), approximately 10 kDa bis(monomalonate) A (2 g, 200 μmol), and the reducing agent dithiothreitol (23.8 mg, 1.54 μmol) were suspended in DMSO (26 mL). The mixture was then heated to 85°C in an oil bath and stirred for 120 minutes using a Hercules Stirrer (trade name: MODEL HERAXLES / 16G, manufactured by Koike Precision Machinery Co., Ltd.), yielding a brown, transparent solution. The solution was applied to a metal plate with a thin release film (Teijin Film Solutions Co., Ltd., thickness 38 μm) using a doctor blade (Imoto Machinery Co., Ltd., application width 80 mm, gap 400 μm), dried at 60°C for at least 2 hours using a constant temperature oven with a blower, and then the solvent was removed using a vacuum oven at 80°C for 15 hours to obtain a brown, transparent film. The structure of bis(monomalonate) A is as follows:

[0150] Protein G (0.52 g, 47.2 μmol), approximately 10 kDa PEG-I (0.48 g, 200 μmol) with the following structure, and the reducing agent sodium cyanoborohydride (59.8 mg, 952 μmol) were suspended in DMSO (17.3 mL). The mixture was then heated to 85°C in an oil bath and stirred for 60 minutes using a Hercules Stirrer (trade name: MODEL HERAXLES / 16G, manufactured by Koike Precision Machinery Co., Ltd.), yielding a clear, brown solution. The solution was applied to a metal plate with a thin release film (manufactured by Teijin Film Solutions Co., Ltd., thickness: 38 μm) using a doctor blade (manufactured by Imoto Manufacturing Co., Ltd., application width: 80 mm, gap: 400 μm). The solution was then dried for at least 2 hours at 60°C using a constant-temperature oven, and the solvent was removed using a vacuum oven at 80°C for 15 hours to yield a clear film. The structure of PEG-I is as follows:

[0151] [Method for Measuring Maximum Elongation] The obtained film was punched into dumbbell shapes 105 mm long and 5 mm wide using a punch (SA-1008, manufactured by Tester Sangyo Co., Ltd.) and stored for at least 24 hours at a room temperature of 20°C and a humidity of 65%. The stored samples were subjected to a tensile test under the same conditions using a tensile tester (AG-X plus 50kN, manufactured by Shimadzu Corporation) with a load cell of 1 kN, a chuck distance of 71.0 mm, and a pulling rate of 200 mm / min. The measurement results are shown in the "Maximum Elongation" column in Figure 1. A larger maximum elongation value indicates that the first and second segments have reacted sufficiently to produce a sufficient amount of block copolymer.

[0152] [Method of Determining Viscosity] The viscosity was determined by the appearance of the solution immediately after synthesis when it was cast into a film. The examples labeled "Rise" were viscous and spindly compared to the examples labeled "No Change," and were clearly different in appearance from the others. A higher viscosity indicates that the first and second segments have reacted sufficiently, resulting in a sufficient amount of block copolymer being obtained.

[0153] Figure 1 shows that the production of proteins D to G resulted in an increase in production yield compared to the production of protein CTR. In Figure 1, protein CTR is labeled "Control," and proteins A to G are labeled "Positions A to G," respectively. Figure 1 shows that, compared to protein CTR, there was no change in viscosity when protein D, in which the N-terminal reactive site was moved within the hydrophobic tag, was used. However, the viscosity increased when proteins F to G, in which the C-terminal reactive site was moved to the C-terminus of the hydrophobic tag or within 9 residues of the C-terminus of the main sequence, were used. Generally, viscosity increases as the reaction progresses. Therefore, it can be said that the reactivity of these molecular candidates was improved by placing the N-terminal reactive site within the main sequence and by moving the C-terminal reactive site to the C-terminus of the hydrophobic tag or within 9 residues of the C-terminus of the main sequence. Furthermore, comparing proteins CTR, E, and F in Figure 1 reveals that the maximum elongation of the resulting film is greater when the most N-terminal reactive site is located within the main sequence and the most C-terminal reactive site is located within the C-terminus of the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence. Furthermore, it can be seen that the maximum elongation of the resulting film tends to increase the closer the N-terminal reactive site is located within the main sequence and the C-terminal reactive site is positioned closer to the C-terminus in the amino acid sequence containing the main sequence and the hydrophobic tag. On the other hand, it can be seen that proteins B to D, in which the most N-terminal reactive site is located within the hydrophobic tag, have a smaller maximum elongation. Furthermore, Figure 1 reveals that proteins B to D, in which the most N-terminal reactive site is moved within the hydrophobic tag, have more disulfide bonds than protein A, while proteins E and F, in which the most C-terminal reactive site is moved within the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence, have fewer disulfide bonds than protein A.

[0154] From the above, it can be seen that a block copolymer using a protein that contains a main sequence and hydrophobic tags at the N-terminus and C-terminus of the main sequence, and has at least two reactive sites that react with the second segment and a compound having a reactive group, and in which the most N-terminal reactive site is located within the main sequence or within the N-terminal hydrophobic tag, and the most C-terminal reactive site is located within the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence, has excellent productivity.

[0155] Example 2: Study on Block Copolymer (BCP) Using Protein G A film containing the block copolymer obtained using the above-mentioned protein G was cut into small pieces and dissolved in HFIP containing TFA salt together with the reducing agent TCEP, followed by GPC. The results shown in Figure 2 were obtained. The dashed line indicates the peak of protein G (PRT3463), and the solid line indicates the peak of the obtained BCP. Furthermore, a BCP using bis(monomalonate) A was produced using the same method as the production of the block copolymer obtained using protein G described above, except that bis(monomalonate) A was used instead of PEG-I and dithiothreitol was used as the reducing agent. In the BCP, the peak of the raw material BP was significantly reduced, and a large peak was observed in the high molecular weight portion not observed in BP. This suggested that PRT3463 and PEG-I had reacted to produce BCP. The obtained film was subjected to a tensile test in the same manner as for the other BCPs. The results are shown in Figure 3. Similar to the BCP using bis(monomalonate) A, a soft and highly elongated film was produced. The obtained film was subjected to a jungle test at a temperature of 60°C and a humidity of 60%, and the film's degradation characteristics were examined. The results are shown in Figure 4. The dotted line shows the results for the BCP using bis(monomalonate) A, and the solid line shows the results for the BCP using PEG-I. It was found that the BCP using PEG-I showed less deterioration in elongation than the BCP using bis(monomalonate) A (film produced using the above-mentioned protein F). Furthermore, the changes in physical properties suggested that the reaction was proceeding via lysine rather than cysteine.

[0156] Example 3: Synthesis of BCP using PCB. A protein (2.25 g, 209 μmol) having the amino acid sequence (PRT3177) represented by SEQ ID NO: 15 and the reducing agent dithiothreitol (193.1 mg, 1.25 μmol) were suspended in DMSO (21.8 mL). The mixture was then heated to 85°C in an oil bath and reduced for 30 minutes using a Hercules stirrer (trade name: MODEL HERAXLES / 16G, manufactured by Koike Precision Machinery Co., Ltd.). Approximately 3 kDa PCB (3.75 g, 375 μmol) was then added, and the mixture was stirred again for 30 minutes in an 85°C oil bath, yielding a brown, transparent solution. The structure of PCB is as follows: The solution was applied to a metal plate with a thin release film (Teijin Film Solutions Co., Ltd., 38 μm thick) using a doctor blade (Imoto Manufacturing Co., Ltd., application width 80 mm, gap 400 μm). The solution was then dried at 60°C for at least 2 hours using a constant temperature oven with a blower. The solvent was then removed using a vacuum oven at 80°C for 15 hours to obtain a transparent film. The obtained film was cut into small pieces and dissolved in HFIP containing TFA salt together with the reducing agent TCEP for GPC. The results are shown in Figure 5. The dashed line indicates the peak of PRT3177, and the solid line indicates the peak of the obtained BCP. The BCP peaks were large in the high molecular weight portion, which was not observed in PRT3177. This suggests that PRT3177 reacted with PCB to produce BCP. The obtained film was subjected to a tensile test in the same manner as for other BCPs. The results are shown in Figure 6. The prepared film was confirmed to be elongated by approximately 300%. Furthermore, block copolymers using PCB have the advantages of improved tensile strength at yield point and reduced water swelling (reduced hydrolysis resistance).

[0157] Example 4: Synthesis of BCP by Mechanochemical Method. 49 g of dried protein (PRT3177, 10,763 g / mol) having the amino acid sequence (PRT3177) represented by SEQ ID NO: 15, 51 g of bis(monomalonate) A (11,000 g / mol) (molar ratio 1:1), and 0.49 g of reducing agent DTT (BP ratio 1 wt%) were thoroughly mixed. 11 mL of DMSO (solid content ratio 12.1 wt%) was added and thoroughly mixed in advance. The mixture was gradually fed at a rate of 288 g / h into a twin-screw extrusion kneader (manufactured by Technovel Co., Ltd.) at 60°C and an extrusion rate of 273 g / h. GPC analysis of the resulting material was performed using the same equipment and method as for the other BCPs. The results shown in Figure 7 were obtained. The dashed line indicates the peak of Protein F (PRT3463), and the solid line indicates the peak of the obtained BCP. As a result of the GPC measurement, a large GPC peak was observed at a position higher in molecular weight than the raw material protein (BP) and PEG, suggesting that BCP synthesis was also progressing when PRT3177 was used.

Claims

1. A block copolymer having a first segment containing a protein structure and one or more second segments containing a molecular group having a plasticizing function for the protein structure and bound to the first segment, wherein the protein structure comprises a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and has at least two reactive sites that react with the second segment and a compound having a reactive group, and the reactive site closest to the N-terminus of the reactive sites is present within the hydrophobic tag on the N-terminus side or is present between the N-terminus of the protein structure and the NH ２ or is present in a main sequence, and the reactive site closest to the C-terminus of the reactive sites is present in a hydrophobic tag on the C-terminus side or within 9 residues from the C-terminus of the main sequence.

2. The block copolymer described in claim 1, wherein the reactive site closest to the C-terminus is located at the C-terminus of the hydrophobic tag on the C-terminus side or within 9 residues from the C-terminus of the main sequence.

3. The block copolymer according to claim 1 or 2, wherein the reactive site closest to the N-terminus is located at the N-terminus of the N-terminal hydrophobic tag or within the main sequence.

4. The block copolymer according to any one of claims 1 to 3, wherein, of the reactive sites, the most N-terminal reactive site present in the main sequence and the most C-terminal reactive site present in the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence are thiol groups.

5. The block copolymer of claim 4, wherein the compound has a thiol-reactive group that reacts with the thiol.

6. The block copolymer according to any one of claims 1 to 3, wherein, of the reactive sites, the most N-terminal reactive site present within the N-terminal hydrophobic tag and the most C-terminal reactive site present within the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence are amino groups.

7. The block copolymer of claim 6, wherein said compound has an amino-reactive group that reacts with said amine.

8. The block copolymer according to any one of claims 1 to 7, wherein the hydrophobic tag is GFIL or two or more repeats of GFIL.

9. The block copolymer according to any one of claims 1 to 8, wherein the molecular weight of the protein structure is 1 to 300 kDa.

10. A method for producing a block copolymer by combining a compound having a first segment containing a protein structure with a compound having a second segment containing a molecular group having a plasticizing function for the protein structure, wherein the protein structure comprises a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and has at least two reactive sites that react with the second segment and a compound having a reactive group, and the reactive site closest to the N-terminus of the reactive sites is present within the hydrophobic tag on the N-terminus side or is present at the N-terminus of the protein structure, NH ２ or is present within a main sequence, and the reactive site closest to the C-terminus of the reactive sites is present within a hydrophobic tag on the C-terminus side or is a protein structure present within 9 residues from the C-terminus of the main sequence.

11. A method for producing the block copolymer according to claim 10, comprising a step of bonding the compound having the first segment and the compound having the second segment by mechanochemical treatment.

12. The method for producing a block copolymer according to claim 10, comprising adding a compound having the first segment and a compound having the second segment to a solvent, wherein the compound having the second segment is added in a non-solution state.

13. The method for producing a block copolymer according to claim 12, comprising adding a reducing agent, a compound having the first segment, and a compound having the second segment to the solvent in this order.

14. An artificial protein comprising a primary sequence and hydrophobic tags attached to the N-terminus and C-terminus of the primary sequence, and also comprising hydrophobic tags at the N-terminus and C-terminus, and having at least two cysteine residues, the most N-terminal cysteine residue of said cysteine residues being present in the primary sequence, and the most C-terminal cysteine residue of said cysteine residues being present in the C-terminal hydrophobic tag or within 9 residues of the C-terminus of the primary sequence.

15. An artificial protein comprising a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence, and also comprising hydrophobic tags at the N-terminus and C-terminus, having at least one lysine residue, wherein the lysine residue closest to the N-terminus is present within the N-terminal hydrophobic tag or there is no lysine residue within the N-terminal hydrophobic tag, and the lysine residue closest to the C-terminus is present within the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence.

16. The artificial protein of claim 14 or 15, wherein the hydrophobic tag is GFIL or two or more repeats of GFIL.

17. The artificial protein according to any one of claims 14 to 16, having a molecular weight of 1 to 300 kDa.

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