Method for producing regenerated polypeptide molded article

Abstract

The present invention addresses the problem of developing a method for suppressing embrittlement of a molded article due to an excessive β-sheet structure while retaining an ɑ-helix during rapid dehydration treatment of a regenerated polypeptide.　An aqueous solution of an ɑ-βt coexisting polypeptide is used as a raw material, and a coagulating liquid used in the rapid dehydration treatment is cooled to a predetermined temperature to coagulate and mold the aqueous solution.

Description

Method for producing regenerated polypeptide molded article

[0001] The present invention relates to a method for producing a regenerated polypeptide molded article, and a regenerated polypeptide molded article obtained by the method.

[0002] Synthetic resins represented by plastics are used in all places of human society and have become indispensable materials for modern life. However, on the other hand, problems of global warming caused by a large amount of carbon dioxide generated by combustion, and problems of environmental pollution caused by waste plastics such as microplastics due to their chemical stability that does not biodegrade have become apparent.

[0003] Therefore, in recent years, toward the realization of a sustainable society, development of environmentally friendly and highly safe alternatives to synthetic resins using fossil fuel as a raw material has been desired. For example, polypeptide molded articles using proteins or the like as a raw material is one of such alternatives.

[0004] Fibroin protein has attracted attention as a raw material for polypeptide molded articles. Fibroin protein is a fiber component of Bombyx mori silk and spider silk (dragline silk), and has a β-sheet structure as a crystal structure, so it exhibits high toughness with an excellent balance between strength and elongation, and since it is composed of a polypeptide, it has both biodegradability and high biocompatibility. Therefore, polypeptide molded articles using fibroin protein as a raw material are expected as materials in a wide range of fields such as medical materials and structural materials.

[0005] Generally, when processing fibroin protein into a polypeptide molded article, liquid protein is used as a raw material. Examples thereof include liquid fibroin protein before fiber formation stored in the silk gland of Bombyx mori or the like (so-called liquid silk), and liquid fibroin protein obtained by expression in Escherichia coli or the like by genetic recombination technology. In addition, an aqueous fibroin protein solution obtained by dissolving already fiber-formed solid fibroin protein in a solvent to form a liquid state can also be used as a raw material. Regenerated fibroin protein obtained by coagulating an aqueous fibroin protein solution and re-forming it into a solid state is expected to be applied in various fields due to its ease of processing and high versatility.

[0006] For regenerated fibroin proteins, an aqueous solution of fibroin H-chain protein from silkworm (Bombyx mori) silk is generally used. However, the polypeptide coagulation molded product obtained by dehydrating this aqueous solution is soluble in water because it contains a water-soluble crystalline structure consisting of a β-turn aggregate called Silk-I type in part of its molecule. Therefore, during molding, insolubilization treatment by rapid dehydration using alcohol or heat treatment is necessary.

[0007] However, this process creates an excessive β-sheet structure within the regenerated fibroin protein molecule, resulting in a hard and brittle texture in the molded product after the process. This is the main reason delaying the practical application of molded products made from regenerated silkworm fibroin H-chain protein in the fields of medical materials and artificial fibers. For example, when used as a medical material, the high β-sheet structure of the regenerated fibroin H-chain protein can lead to problems such as increased thrombosis and decreased enzymatic degradability (Non-Patent Literature 1, 2).

[0008] On the other hand, it is also possible to produce molded articles of regenerated fibroin H-chain protein from aqueous solutions of fibroin H-chain protein from silkworms (Samia cynthia ricini) and spiders. However, since the polypeptide coagulated molded articles obtained by dehydrating these aqueous solutions contain α-helices, they are soluble in water, similar to silkworms, and therefore insolubilization treatment is essential during molding, resulting in a hard and brittle physical property.

[0009] To solve the above problems, the inventors conducted extensive research and found that fibroin H-chain protein derived from bagworms forms α-helices and β-turns in aqueous solution, and that in the polypeptide coagulated molded product obtained by dehydrating the aqueous solution, it has an amino acid sequence that forms α-helices and β-sheet structures. Therefore, it was found that the coagulated molded product of bagworm fibroin H-chain protein is insoluble in water without undergoing insolubilization treatment, is highly ductile, flexible, and has appropriate strength, thus solving the above problems.

[0010] However, in thin-film molding, microparticle molding, or fiber molding, rapid solidification is required during processing, making rapid dehydration treatment using alcohol or the like essential. However, this treatment re-emerges the aforementioned embrittlement problem, and also causes the material to break at low stretch ratios during stretching. These are particularly serious problems in fiber molding and are the main reason why the practical application of regenerated fibroin protein has not yet been realized.

[0011] Kim HH, et al., 2016, Polymer, 90: 26-33.Guo C., , et al.,2020, Biomacromolecules, 21: 1678-1686.

[0012] When manufacturing molded polypeptide articles from aqueous solutions of α-βt / s coexisting polypeptides, such as the bagworm fibroin H-chain protein, rapid dehydration during molding can induce the crystallization of excessive β-sheet structures within the polypeptide molecule, resulting in the loss of α-helices and ultimately causing the molded article to become brittle.

[0013] The present invention aims to develop a method for producing regenerated polypeptides using an aqueous solution of α-βt-coexisting polypeptides as a raw material, which retains α-helices and suppresses the embrittlement of molded articles due to excessive β-sheet structures.

[0014] To solve the above problems, the inventors conducted extensive research and discovered that by cooling a coagulation solution such as alcohol used in the production of regenerated polypeptide molded articles to a predetermined temperature, coagulation can be performed, and then molding can be carried out, thereby enabling the production of regenerated polypeptide molded articles that retain α-helices. This is presumed to be due to the synergistic effect of the coagulation induction ability of the coagulation solution and the induction of coagulation and molding based on the viscosity increase of fibroin protein induced by low temperature, which enabled coagulation and molding even when using a low-concentration coagulation solution that would not normally coagulate and processing for a short time. Furthermore, when the polypeptide is fibroin H-chain protein, high stretching exceeding 100% of the stretching strain became possible in the stretching process of the regenerated fibroin H-chain protein. The present invention is based on the results of the above research and development and includes the following.

[0015] (1) A method for producing a recycled polypeptide molded article, comprising a coagulation step of coagulating an aqueous solution of an α-βt / s coexisting polypeptide having one or more α-helix-forming sequences and β-turn-forming sequences in one molecule, by contact with a coagulation solution, wherein the α-helix-forming sequence consists of an amino acid sequence that forms an α-helix in whole or in part in the aqueous solution or the polypeptide coagulated molded article, the β-turn-forming sequence consists of an amino acid sequence that forms a β-turn in whole or in part in the aqueous solution and forms a β-sheet structure in whole or in part in the polypeptide coagulated molded article, and the temperature of the coagulation solution in the coagulation step is -3°C or higher and below the inflection temperature in the temperature-viscosity correlation curve. (2) The method according to (1), further comprising a dissolution step of dissolving the α-βt / s coexisting polypeptide in water before the coagulation step to prepare an aqueous solution of the α-βt coexisting polypeptide. (3) The method according to (1) or (2), further comprising a stretching step of stretching the recycled polypeptide molded article obtained after the coagulation step. (4) The method for producing an amino acid sequence according to any one of (1) to (3), wherein the α-helix-forming sequence consists of the amino acid sequence shown in SEQ ID NO: 1 and / or GGX (where X is A or S) or the amino acid sequence shown in SEQ ID NO: 2. (5) The method for producing an amino acid sequence according to any one of (1) to (4), wherein the β-turn-forming sequence consists of the amino acid sequence shown in GXGXGX (where X is A or S) or SEQ ID NO: 3 and / or the amino acid sequence shown in SEQ ID NO: 4. (6) The method for producing an amino acid sequence according to any one of (1) to (5), wherein the α-βt / s coexisting polypeptide contains an α-βt / s coexisting fibroin H chain protein. (7) The method for producing an amino acid sequence according to (6), wherein the α-βt / s coexisting fibroin H chain protein contains a mutant fibroin H chain protein. (8) The method for producing an amino acid sequence according to (7), wherein the α-βt / s coexisting fibroin H chain protein is derived from bagworm silk. (9) The method for producing an amino acid sequence according to any one of (1) to (8), wherein the coagulation solution is a solution containing a lower alcohol. (10) The method for producing the lower alcohol according to (9), wherein the lower alcohol concentration in the solution containing the lower alcohol is 10% to 90%. (11) The method for producing the lower alcohol according to (10), wherein the inflection temperature is 6°C to 7°C. (12) A molded article of a regenerated polypeptide containing one or more α-helix structures in the molecule.(13) The regenerated polypeptide molded article according to (12), wherein the aqueous protein solution contains fibroin H chain protein. (14) The regenerated polypeptide molded article according to (13), wherein it is a thin film molded article, a fine particle molded article, or a fiber molded article.

[0016] According to the method for producing recycled polypeptide molded articles of the present invention, even in a polypeptide insolubilized molded article that has undergone insolubilization treatment, it is possible to produce a recycled polypeptide molded article that retains α-helices and does not undergo embrittlement.

[0017] This figure shows the wide-angle X-ray diffraction results of polypeptide solidified molded products obtained by freeze-drying an aqueous solution of natural bagworm FibH. This figure compares the wide-angle X-ray diffraction results of regenerated bagworm FibH solidified molded products obtained by molding an aqueous solution of natural bagworm FibH in solidifying solutions at different temperatures. This is a plot showing the relationship between viscosity and temperature of an aqueous solution of natural bagworm FibH. In the figure, the temperatures indicated by the arrows are such that smaller values ​​represent the lower limit of the inflection temperature, and larger values ​​represent the upper limit of the solidifying solution. This is a plot showing the relationship between viscosity and temperature of an aqueous solution of mutant bagworm FibH. In the figure, the temperatures indicated by the arrows are such that smaller values ​​represent the lower limit of the inflection temperature, and larger values ​​represent the upper limit of the solidifying solution.

[0018] 1. Method for producing molded articles of recycled polypeptide 1-1. Overview The first aspect of the present invention is a method for producing molded articles of recycled polypeptide. The production method of the present invention is characterized by contacting an aqueous solution of α-βt-containing polypeptide with a coagulation solution cooled to a predetermined temperature to coagulate it.

[0019] According to the manufacturing method of the present invention, even when an aqueous solution of α-βt-containing polypeptide is coagulated and molded, a molded article of recycled polypeptide can be obtained that has one or more α-helices in the molecule, is not embrittlement, and has high ductility.

[0020] 1-2. Definitions The following terms used herein are defined below.

[0021] In this specification, "molded article" refers to a solid body that has acquired a certain hardness and a specific shape. It is preferable that it has undergone a molding process, but it may also be in an unmolded state. "Solid body" refers to a solid substance other than a liquid or gas. In this specification, semi-solid substances such as gels are also included.

[0022] In this specification, "polypeptide molded article" means a molded article containing a polypeptide such as a protein as its main component. It may also consist solely of polypeptides. Examples include thin film molded articles, fine particle molded articles, or fiber molded articles. In this specification, unless otherwise specified, "polypeptide molded article" means a molded article of recycled polypeptide. Furthermore, when simply referred to as a polypeptide molded article in this specification, it shall include both polypeptide coagulated molded articles and polypeptide insolubilized molded articles, which will be described later.

[0023] In this specification, "regenerated polypeptide" refers to a solid polypeptide obtained by coagulating a liquid polypeptide that was dissolved in an aqueous solution. In this specification, this refers to regenerated fibrous protein regenerated from an aqueous solution of fibrous protein, and in particular, regenerated fibroin H-chain protein regenerated from an aqueous solution of fibroin H-chain protein.

[0024] In this specification, "recycled polypeptide molded article" refers to a product obtained by molding a recycled polypeptide to have a certain hardness and a specific shape. In this specification, polypeptide coagulation molded articles and polypeptide immobilization molded articles are included.

[0025] In this specification, "polypeptide coagulated molded article" refers to a regenerated polypeptide molded article obtained by coagulating an aqueous polypeptide solution, for example, by dehydration, without insolubilization or other treatment. In this specification, substantially, it means a polypeptide coagulated molded article consisting of a polypeptide that contains one or more α-helix-forming sequences and β-turn-forming sequences in its molecule, i.e., an α-βs coexisting polypeptide coagulated molded article (α-helix-β-sheet coexisting coagulated molded article).

[0026] In this specification, "polypeptide-insolubilized molded article" refers to a regenerated polypeptide molded article that has been immobilized by solidifying an aqueous polypeptide solution, for example, by rapid dehydration. Polypeptide-insolubilized molded articles typically refer to βs polypeptide molded articles that have become insoluble in water because the immobilization treatment induces most of the α-helices or β-turns within the polypeptide to crystallize into β-sheet structures.

[0027] In this specification, "solidification" means the process by which a liquid becomes a solid, i.e., solidification.

[0028] In this specification, "insolubilization treatment" refers to a treatment that insolubilizes a regenerated polypeptide. This treatment induces the crystallization of β-sheet structures within the polypeptide, resulting in the formation of excessive β-sheet structures within the molecule, thus making the regenerated polypeptide insoluble in water. Specific examples of insolubilization treatments, though not limited to these, include heat treatment, alcohol treatment outside the temperature range of the present invention, stretching treatment, or combinations thereof. While rapid dehydration using a coagulation solution such as alcohol usually induces excessive β-sheet structure formation within the molecule as described above and therefore constitutes an insolubilization treatment, the method using a coagulation solution in the production method of the present invention does not constitute an insolubilization treatment because the coagulation solution is performed at a low temperature, thereby suppressing excessive β-sheet structure formation.

[0029] In this specification, "dehydration treatment" refers to a treatment in which an aqueous solution of α-βt-containing polypeptide is dehydrated using a coagulation solution.

[0030] In this specification, "rapid dehydration" means dehydrating rapidly in a short amount of time.

[0031] In this specification, "silk thread" refers to thread derived from insects, primarily composed of fibrous proteins. However, this specification also includes threads derived from arthropods such as spider silk. Silk is spun by insect larvae and adults for purposes such as nesting, movement, anchoring, cocooning, and foraging. In this specification, when simply referred to as "silk thread," it generally means a broad, general type of silk thread without specifying the insect of origin. When referring to silk thread derived from a specific insect, the name of the organism of origin, such as silkworm silk or bagworm silk, shall be placed before "silk thread."

[0032] In this specification, "fibrous protein" refers to a protein having a fibrous form. Examples include keratin protein, which makes up hair, or fibroin protein, which makes up silk thread.

[0033] In this specification, "fibroin protein" refers to the silk protein that constitutes silk threads derived from insects. Unless otherwise specified, when "fibroin protein" is used in this specification, it refers to fibroin H-chain protein.

[0034] In this specification, "fibroin H-chain protein" (often abbreviated as "FibH") refers to the main protein constituting the fibroin complex (silk fibroin elementary unit; SFEU complex), which is the fibrous protein component of silk thread. Generally, FibH refers to the high molecular weight fibrous protein that constitutes silk thread derived from insects such as silkworms and bagworms. However, in this specification, spidoin protein, a high molecular weight fibrous protein that constitutes silk thread, particularly the dragline silk, derived from organisms of the order Araneae or Acari, is also included in the definition of FibH in the following description. FibH usually has an amino acid sequence consisting of repeating clusters of glycine residues (G) and alanine residues (A). In this specification, FibH may be natural FibH or artificial FibH. In this specification, FibH refers to β-fibroin, which takes the β-sheet structure as the crystalline structure of natural silk thread.

[0035] In this specification, "natural fibroin H-chain protein" (natural FibH) refers to FibH that exists in nature. Natural FibH is defined as a protein whose full-length amino acid sequence is identical to that of naturally occurring FibH. Since natural FibH is the protein encoded by the wild-type FibH gene in various organisms, it is often referred to as "wild-type FibH" in this specification.

[0036] As used herein, the term "artificial fibroin heavy chain protein" (artificial FibH) refers to FibH that does not exist in nature. It is a fibroin heavy chain protein that is artificially modified in all or part of natural FibH by genetic recombination technology or the like.

[0037] As used herein, the term "mutant fibroin heavy chain protein" (often referred to as "mutant FibH" herein) refers to FibH composed of an amino acid sequence different from that of wild-type FibH, and is synonymous with the aforementioned artificial FibH in the present specification. As FibH, it does not matter whether it has physical properties equivalent to those of wild-type FibH or not. Examples of mutant FibH include mutant FibH obtained by introducing addition, deletion, and / or substitution of one or more amino acids into the amino acid sequence of FibH, and chimeric FibH (hybrid FibH) obtained by fusing amino acid sequences of FibH derived from two or more different insects.

[0038] As used herein, the term "regenerated fibroin heavy chain protein" (regenerated FibH) refers to a product in which FibH dissolved in a solvent and turned into a liquid state is converted into a solid state having a specific shape again by coagulation.

[0039] As used herein, "α-helix" is one of the secondary structures in a polypeptide, which forms a right-handed helical structure. The pitch per helix is 0.54 nm, a hydrogen bond is formed between the imino group of the 1st amino acid residue and the carbonyl group of the 4th amino acid residue among four consecutive amino acid residues of the peptide chain, and contains 3.6 amino acid residues per rotation.

[0040] As used herein, "β-turn" is one of the secondary structures in a polypeptide, in which a single molecular chain bends and forms a sheet structure through intramolecular hydrogen bonding.

[0041] As used herein, "β-sheet structure" is one of the secondary structures in a polypeptide, in which at least 2 to 3 adjacent parallel β-strands are laterally linked by intermolecular hydrogen bonds to form a pleated sheet structure that is formed by overall twisting. A substance having a β-sheet structure in the molecule may be insoluble in water.

[0042] As used herein, unless otherwise specified, the term "one molecule" refers to one molecule of FibH, that is, FibH consisting of a single peptide chain.

[0043] As used herein, the term "plurality" refers to, for example, 2 to 1000, 2 to 800, 2 to 600, 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 150, 2 to 100, 2 to 80, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0044] As used herein, an "α-βt / s coexisting polypeptide" (α-helix-β-turn / sheet coexisting polypeptide) is a polypeptide characterized by containing one or more α-helix forming sequences and one or more β-turn forming sequences respectively in one molecule of the polypeptide. Due to this feature, the α-βt / s coexisting polypeptide has the property of allowing α-helix and β-turn to coexist as stable molecular conformations in the peptide chain in an aqueous solution, and allowing α-helix and β-sheet structures to coexist as metastable molecular conformations in the peptide chain in a solidified molded product of the polypeptide. An example of the α-βt / s coexisting polypeptide includes a case where the polypeptide is a fibrous protein, that is, an α-βt / s coexisting fibrous protein. Further, as a specific example of the α-βt / s coexisting fibrous protein, there can be mentioned a case where the fibrous protein is fibroin heavy chain protein, that is, an α-βt / s coexisting fibroin heavy chain protein. The specific configurations of this α-βt / s coexisting fibroin heavy chain protein and an aqueous solution thereof will be described later.

[0045] An α-βt / s coexisting polypeptide can have at least one α-helix-forming sequence and one β-turn-forming sequence in its amino acid sequence, regardless of their position or number. For example, in the amino acid sequence of an α-βt / s coexisting polypeptide, the α-helix-forming sequences and β-turn-forming sequences may be arranged in separate, consecutive clusters, or they may be arranged in alternating consecutive clusters. Furthermore, the α-helix-forming sequences and β-turn-forming sequences may be randomly dispersed within the amino acid sequence of an α-βt / s coexisting polypeptide.

[0046] In this specification, "aqueous aqueous solution of α-βt-coexisting polypeptide" (aqueous aqueous solution of α-helix-β-turn-coexisting polypeptide) is an aqueous solution in which α-βt / s-coexisting polypeptide is dissolved. As described above, in the aqueous solution, the α-βt / s-coexisting polypeptide has α-helices and β-turns coexisting in a stable molecular form within the peptide chain. The α-βt / s-coexisting polypeptide contained in the aqueous solution of α-βt-coexisting polypeptide may be of the same type or of different types.

[0047] In this specification, "aqueous solution" refers to a solution which is a liquid mixture composed of two or more substances, with water as the primary solvent. The solute dissolved in the aqueous solution may be a solid, liquid, or gas. Examples of solutes include electrolytes and water-soluble substances. Liquid solutes may also include organic solvents such as lower alcohols.

[0048] In this specification, "α-helix-forming sequence" refers to an amino acid sequence that, in whole or in part, forms an α-helix in an aqueous polypeptide solution containing α-βt or in a polypeptide coagulated molded article. For example, an amino acid sequence represented by (A)n (an integer n ≥ 6) is an example, specifically the amino acid sequence shown in Sequence ID No. 1. Another example is an amino acid sequence represented by (GGX)n (where X is an alanine residue (A) or a tyrosine residue (Y), and n ≥ 1) is an example, specifically the amino acid sequence shown in Sequence ID No. 2. The helix formed by (GGX)n corresponds to a "31-helix," which has a helix pitch slightly shorter than that of an α-helix, but in this specification, the "31-helix" is also included as a structure of the α-helix.

[0049] In this specification, "β-turn-forming sequence" refers to an amino acid sequence that, in whole or in part, forms a β-turn in an aqueous polypeptide solution containing α-βt, and in whole or in part, forms a β-sheet structure in a polypeptide coagulated molded product. Thus, this β-turn-forming sequence can form different secondary structures in aqueous solution and in a polypeptide coagulated molded product. Examples of amino acid sequences exhibiting such characteristics include the amino acid sequence represented by (GX)n (where X is an alanine residue or a serine residue, and n is an integer ≥ 3), specifically the amino acid sequence shown in Sequence ID No. 3. Another example is the amino acid sequence represented by (GAGAGX)n (where X is a serine residue or a tyrosine residue, and n is an integer ≥ 1), specifically the amino acid sequence shown in Sequence ID No. 4. However, exceptionally, in the wild-type silkworm fibroin H-chain protein, the (GX)n and (GAGAGX)n sequences do not form a β-sheet structure in the polypeptide coagulated molded product, but instead form a water-soluble crystalline structure consisting of an aggregate of β-turns called the Silk-I type.

[0050] In this specification, "viscosity" refers to the degree of stickiness of a fluid, also known as viscosity coefficient. It is expressed in SI units as Pa·s. Viscosity is temperature-dependent; in the case of liquids, viscosity generally decreases as the temperature rises. In this specification, the viscosity coefficient refers to the viscosity of an aqueous solution of α-βt-containing polypeptide.

[0051] In this specification, "inflection temperature" means the temperature at which an inflection point occurs in the correlation curve between temperature and viscosity. "Inflection point" means the point at which the curvature of the correlation curve changes sign or becomes stationary.

[0052] In this specification, "thin film molded article" refers to a molded article in the form of a film or sheet. In particular, thin film molded articles of polypeptide solidification are given as examples in this specification.

[0053] In this specification, "microparticle molded articles" refers to molded articles in the form of fine particles, such as beads. In particular, this specification gives examples of microparticle molded articles made from polypeptides.

[0054] In this specification, "fiber molded article" means a fibrous molded article obtained artificially.

[0055] 1-3. Method The manufacturing method of the present invention includes a solidification step as an essential step, and a dissolution step and a stretching step as optional steps. The configuration of each step and the elements used therein will be described below.

[0056] (1) Dissolution step The "dissolution step" is a step in which the α-βt / s coexisting polypeptide is dissolved in water to prepare an aqueous solution of the α-βt coexisting polypeptide to be used in the coagulation step described below. This is an optional step in the production method of the present invention and may be performed as needed, for example, if an aqueous solution of the α-βt coexisting polypeptide has not been prepared.

[0057] The α-βt / s-coexisting polypeptide dissolved in this process may be either a natural α-βt / s-coexisting polypeptide or an artificial α-βt / s-coexisting polypeptide, and may be one or more types. Furthermore, other solutes may be dissolved in water along with the α-βt / s-coexisting polypeptide.

[0058] The α-βt / s coexisting polypeptide used in this process is not limited, but α-βt / s coexisting fibroin H chain protein (often referred to as "α-βt / s coexisting FibH" in this specification) is preferred. The FibH in α-βt / s coexisting FibH may be natural or artificial.

[0059] An example of a natural fibH having an α-βt / s coexisting fibH structure is the polypeptide of the bagworm (often referred to as "bagworm fibH" in this specification). While many natural fibHs, such as silkworm fibH, Eri silkworm fibH, and spider fibH, do not have an α-βt / s coexisting structure, bagworm fibH is one of the few natural fibHs that possesses the characteristics of an α-βt / s coexisting fibH.

[0060] In this specification, "bagworm" refers to the larvae of moths belonging to the family Psychidae, order Lepidoptera. The types of bagworms referred to in this specification are not limited. For example, there are genera such as Acanthopsyche, Anatolopsyche, Bacotia, Bambalina, Canephora, Chalioides, Dahlica, Diplodoma, Eumeta, Eumasia, Kozhantshikovia, Mahasena, Nipponopsyche, Paranarychia, Proutia, Psyche, Pteroma, Siederia, Striglocyrbasia, Taleporia, Theriodopteryx, and Trigonodoma, but the bagworms referred to in this specification may belong to any of these genera. Specific examples of bagworm species include Eumeta japonica and Eumeta minuscula.

[0061] Generally, natural fibH consists of an N-terminal region, a central region, and a C-terminal region as its basic constituent elements, in that order from the N-terminus.

[0062] The "N-terminal region" refers to the region in the amino acid sequence that constitutes natural FibH that is located on the N-terminal side of the central region described below, does not contain repeating units, and consists of 70 to 250 amino acid residues.

[0063] Furthermore, the "central region" is the main region that exhibits the physical properties of natural FibH. This region is responsible for the fibrous properties of FibH and is composed of multiple repeating units consisting of the same and / or different amino acid sequences linked together. A "repeating unit" is the main constituent unit of the central region, and one repeating unit can contain multiple glycine (G) and alanine (A) molecules. Therefore, more than 70% of the amino acid sequence of the central region is composed of glycine and alanine.

[0064] Furthermore, the "C-terminal region" refers to the region in the amino acid sequence that constitutes natural FibH that is located on the C-terminal side of the central region, does not contain repeating units, and consists of 30 to 60 amino acid residues.

[0065] In natural FibH, the α-helix-forming sequence and the β-turn-forming sequence may be contained within the repeating unit of the central region, or they may be contained outside of the repeating unit, such as the N-terminal region and / or the C-terminal region.

[0066] An example of artificial FibH having a structure in which α-βt / s coexist is mutant bagworm FibH.

[0067] In this specification, "mutant bagworm FibH" refers to a mutant FibH based on bagworm FibH, which is a mutant FibH obtained by introducing amino acid mutations into the amino acid sequence of wild-type bagworm FibH, and / or a chimeric FibH obtained by fusing the amino acid sequences of two or more different insects, including bagworms, and which has the structure of α-βt / s coexisting FibH.

[0068] Mutant bagworm FibH contains one or more repeat units in its amino acid sequence. Examples of specific amino acid sequences that constitute one repeat unit in bagworm FibH include the amino acid sequences shown in SEQ ID NOs. 5-13, amino acid sequences in which one or more amino acids are added, deleted, or substituted in the amino acid sequences shown in SEQ ID NOs. 5-13, or amino acid sequences that have 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the amino acid sequences shown in SEQ ID NOs. 5-13 are amino acid sequences of repeat units unique to wild-type bagworm FibH of the giant bagworm moth, which has the α-βt / s coexisting FibH configuration.

[0069] In this specification, "amino acid identity" refers to the percentage of identical amino acids in one polypeptide relative to the total number of amino acids in the other polypeptide, after the amino acid sequences of two polypeptides have been aligned and, if necessary, gaps have been introduced into either amino acid sequence to maximize the degree of amino acid agreement between the two polypeptides. This percentage of amino acid identity can be easily determined using known programs such as the homology search program BLAST (Basic local alignment search tool; Altschul, SF et al, J. Mol. Biol., 215, 403-410, 1990).

[0070] The mutant bagworm FibH may include the amino acid sequence shown in SEQ ID NO: 14, which contains four repeat units consisting of the amino acid sequences shown in SEQ ID NOs: 5 to 13; the amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 14; or the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 14. The amino acid sequence shown in SEQ ID NO: 14 is an amino acid sequence consisting of a part of the wild-type bagworm FibH of the giant bagworm moth.

[0071] An example of a specific base sequence that encodes the amino acid sequence shown in Sequence ID No. 14 is the base sequence shown in Sequence ID No. 15.

[0072] Another example of a mutant bagworm FibH is a terminal-region mutant bagworm FibH, which contains mutations in either the N-terminal region, the C-terminal region, or both of the wild-type bagworm FibH.

[0073] The aforementioned mutation, if it is a deletion, may involve the complete deletion of either the N-terminal region or the C-terminal region, or both, or a partial deletion. Here, "partial" refers to one or two or more consecutive or discontinuous amino acids from the amino acid sequence constituting a specific amino acid region (here, the N-terminal region and / or C-terminal region), and less than the total number of amino acids. Preferably, this corresponds to the number of amino acids that may cause loss of function in the terminal region. For example, this could include 5, 8, 10, 12, 15, 18, or 20 or more consecutive or discontinuous amino acids. In the terminal region mutant bagworm FibH, either the N-terminal region or the C-terminal region, or both, may be deleted.

[0074] In addition to deletions, mutations may also include the addition or substitution of one or more amino acids. The amino acids to be added or substituted are not particularly limited, but amino acids that may lose the function of their terminal region are preferred.

[0075] Specific examples of mutant bagworm FibH include the amino acid sequences shown in SEQ ID NOs: 16-18. SEQ ID NO: 16 is the amino acid sequence of mutant bagworm FibHΔC, which has the amino acid sequence shown in SEQ ID NO: 14, but with the entire C-terminal region deleted. SEQ ID NO: 17 is the amino acid sequence of mutant bagworm FibHΔN, which has the amino acid sequence shown in SEQ ID NO: 14, but with the entire N-terminal region deleted. SEQ ID NO: 18 is the amino acid sequence of mutant bagworm FibHΔN / C, which has the amino acid sequence shown in SEQ ID NO: 14, but with both the N-terminal and C-terminal regions completely deleted.

[0076] Examples of specific base sequences of genes encoding terminal region mutant bagworm FibH include the mutant bagworm FibHΔC gene shown in SEQ ID NO: 19, the mutant bagworm FibHΔN gene shown in SEQ ID NO: 20, and the mutant bagworm FibHΔN / C gene shown in SEQ ID NO: 21.

[0077] The aforementioned mutant bagworm FibH may be a chimeric FibH of bagworm-derived FibH and FibH derived from another insect. For example, a chimeric FibH of bagworm FibH and silkworm FibH can be cited. Specifically, for example, a chimeric FibH of the giant bagworm FibH and silkworm FibH consisting of the amino acid sequence shown in SEQ ID NO: 22 can be cited. In this chimeric FibH, positions 1 to 153 and 466 to 524 are amino acid sequences derived from silkworm FibH, and positions 156 to 463 contain amino acid sequences derived from the giant bagworm FibH. In the case of a chimeric FibH, it may possess the physical properties of the FibH of each of the organisms from which it is derived. Furthermore, it may also be an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 22. An example of a specific base sequence that encodes the amino acid sequence shown in Sequence ID No. 22 is the base sequence shown in Sequence ID No. 23.

[0078] In mutant bagworm FibH, the α-helix-forming sequence and the β-turn-forming sequence may be amino acid sequences present in natural FibH, or they may be amino acid sequences inserted into bagworm FibH in any number during modification using genetic engineering or other technologies. In this case, the properties of the polypeptide molded product obtained by molding the aqueous solution of the mutant bagworm FibH can be controlled by adjusting the respective ratios of the inserted α-helix-forming sequence and the β-turn-forming sequence.

[0079] Mutant bagworm FibH may optionally have an exogenous signal peptide at its N-terminus. A "signal peptide" is an extracellular transit signal necessary for the secretion of proteins biosynthesized by gene expression into the extracellular space. After translation, the signal peptide is cleaved and removed by a signal peptidase before being secreted into the extracellular space. The signal peptide has a positively charged amino acid such as Lys or Arg at its N-terminus, followed by a highly hydrophobic amino acid sequence such as Ala, Leu, Val, Ile, and Phe. Secretory proteins usually have an endogenous signal peptide at their N-terminus. In the case of chimeric FibH of bagworm FibH and silkworm FibH, which is composed of the amino acid sequence shown in Sequence ID No. 22, the amino acids from positions 1 to 21 correspond to the endogenous signal peptide derived from silkworm FibH, so an exogenous signal peptide is not necessary. Furthermore, the C-terminal side of the signal peptide may also have an amino acid sequence containing a post-insertion signal sequence that promotes cleavage and secretion of the signal peptide and / or a signal peptidase recognition site that cleaves the signal peptide from the fusion protein. The amino acid sequence of the signal peptide is not particularly limited. Usually, it is sufficient if it is within the range of 3 to 60 amino acids. The signal peptide DNA encoding the signal peptide is not limited, but examples include signal peptide DNA encoding the silkworm sericin 1 signal peptide containing the amino acid sequence shown in SEQ ID NO: 24 (e.g., DNA containing the base sequence shown in SEQ ID NO: 25), signal peptide DNA encoding the silkworm sericin 2 signal peptide containing the amino acid sequence shown in SEQ ID NO: 26 (e.g., DNA containing the base sequence shown in SEQ ID NO: 27), and signal peptide DNA encoding the silkworm sericin 3 signal peptide containing the amino acid sequence shown in SEQ ID NO: 28 (e.g., DNA containing the base sequence shown in SEQ ID NO: 29).

[0080] The preparation of FibH aqueous solution can be carried out using methods known in the art. For example, it can be carried out according to the methods of Srisuwan and Sihanam (Srisuwan Y., Sihanam P., 2009, J. Appl. Sci., 9: 978-982) or Guo et al. (Guo Y., et al., 2017, Mater. Res. Express, 4, 105404). Typically, FibH is dissolved in a high-salt solution and then desalted to prepare the solution.

[0081] (2) Coagulation Step The "coagulation step" is a step in which an aqueous solution of α-βt-coexisting polypeptide is brought into contact with a coagulation solution at a predetermined temperature to coagulate the α-βt-coexisting polypeptide in the aqueous solution. This is the most distinctive and essential step in the manufacturing method of the present invention, and a molded article of recycled polypeptide can be obtained through this step.

[0082] In this specification, "coagulation solution" refers to a liquid that has the effect of coagulating an α-βt / s coexisting polypeptide dissolved in an aqueous solution of an α-βt coexisting polypeptide. The coagulation solution may be any liquid based on any action, and is not limited to those that, by coagulation, cause all or part of the α-helix-forming sequences in the aqueous solution of the α-βt coexisting polypeptide to form α-helices, and all or part of the β-turn-forming sequences to form β-sheet structures. For example, a coagulation solution that removes water from the aqueous solution of the α-βt coexisting polypeptide, i.e., dehydrates it, is an example. Examples of coagulation solutions having such a dehydrating effect include lower alcohols having 1 to 6 carbon atoms (methanol, ethanol, n-propanol, i-propanol, t-butanol, etc.), polyhydric alcohols (glycerin, ethylene glycol, polyethylene glycol, etc.), acetone, dimethyl sulfoxide, tetrahydrofuran, diethyl ether, acetonitrile, N,N-dimethylformamide, N-methyl-2-pyrrolidone, 1,4-dioxane, or solutions containing the same. If the coagulation solution is a solution containing lower alcohols or acetone, its concentration may be, for example, 10% to 98%, 20% to 97%, 30% to 90%, 30% to 96%, 40% to 95%, 50% to 90%, 60% to 85%, or 70% to 80%.

[0083] The coagulation solution used in this process is preferably at a predetermined temperature. In particular, if it has a dehydrating effect, it must be at a predetermined temperature. The "predetermined temperature" in this process is -3°C or higher, -2°C or higher, -1°C or higher, 0°C or higher, 1°C or higher, or 2°C or higher, and below the inflection temperature in the temperature-viscosity correlation curve. The inflection temperature in the temperature-viscosity correlation curve can be appropriately determined depending on the type of coagulation solution used in this process. For example, if the coagulation solution is a lower alcohol, the inflection temperature will be 6°C to 7°C. By coagulating in this low temperature range, even if rapid coagulation is performed, the formation of excessive β-sheet structures within the molecule is suppressed, and an α-βs-coexisting polypeptide molded product with residual α-helices can be obtained.

[0084] In this process, the α-βt-coexisting polypeptide aqueous solution is brought into contact with a coagulation solution to coagulate it. The method of contact between the α-βt-coexisting polypeptide aqueous solution and the coagulation solution is not limited, but since rapid coagulation is required, a method of exposing the α-βt-coexisting polypeptide aqueous solution to the coagulation solution is preferred. For example, this can be done by extruding the α-βt-coexisting polypeptide aqueous solution from a nozzle (lip) in a cooled coagulation solution tank, or by suspension coagulation, in which the α-βt-coexisting polypeptide aqueous solution is vigorously stirred in a cooled coagulation solution tank to disperse and suspend it and form particles. Specific examples of extrusion molding methods include wet fiber molding, wet film formation, and wet droplet formation.

[0085] The "wet spinning method" is a method of forming fibers by extruding a solution containing dissolved polymers (in this specification, an aqueous solution of α-βt-containing polypeptides) through the pores of a nozzle (spinneret) into a solidifying solution. This method is suitable when processing regenerated polypeptides into fibers.

[0086] The "wet film formation method" is a method of forming a sheet by extruding a solution containing a dissolved polymer (in this specification, an aqueous solution of α-βt-containing polypeptide) into a solidifying solution through a slit-shaped nozzle (spinaret). This method is suitable when forming regenerated polypeptide into a film.

[0087] The "wet droplet method" is a method of forming particles by injecting a solution containing a dissolved polymer (in this specification, an aqueous solution of α-βt-containing polypeptide) into a solidifying solution through the pores of a nozzle (spinneret). This method is suitable when processing regenerated polypeptides into (fine) particles.

[0088] (3) Stretching step The "stretching step" is a step of stretching the regenerated polypeptide molded product obtained after the solidification step. In the manufacturing method of the present invention, this is an optional step and may be performed as needed.

[0089] In this specification, "stretching" refers to stretching a regenerated polypeptide molded product (such as a thin film molded product or a fiber molded product) made from an α-βs-coexisting polypeptide solidified molded product formed in the solidification process, thereby changing the orientation and crystallization state of the peptide chains in the α-βs-coexisting polypeptide molded product. This process can impart strength and elongation to the regenerated polypeptide molded product. Furthermore, if the solidified molded product before this process is soluble in water, this process can impart insolubility.

[0090] The stretching method involves, for example, performing a stretching operation between multiple rolls with different surface speeds after extrusion molding in the solidification process. Heat treatment may also be performed simultaneously. Examples of heating methods include heating the rolls themselves, using a humid heat chamber filled with high-temperature steam, using a high-temperature liquid bath such as an oil bath, or using a flat or pin-shaped contact heater. The stretching speed, stretching ratio, and heating temperature can be appropriately determined according to the purpose.

[0091] Examples of stretch ratios include 1.5 times (stretch strain of 150%: the same applies below), 1.8 times, 2.0 times, 2.2 times, 2.4 times, 2.6 times, 2.8 times, 3.0 times, 3.2 times, 3.4 times, 3.6 times, 3.8 times, 4.0 times, 4.2 times, 4.4 times, 4.6 times, 4.8 times, or 5.0 times.

[0092] Examples of heating temperatures include 60°C to 220°C, 90°C to 180°C, 100°C to 160°C, 120°C to 150°C, or 130°C to 140°C.

[0093] The recycled polypeptide molded product obtained as a material by the above solidification or stretching process can be further molded into a three-dimensional shape or the like. For example, it can also be molded using vacuum forming or press forming methods using a sheet-like recycled α-βs-coexisting polypeptide molded product.

[0094] 2. Regenerated Polypeptide Molded Articles 2-1. Overview The second aspect of the present invention is a regenerated polypeptide molded article. The regenerated polypeptide molded article of the present invention is characterized by containing one or more α-helix structures in the molecule. One form of a regenerated polypeptide molded article having such characteristics is the regenerated polypeptide molded article obtained by the manufacturing method of the first aspect.

[0095] The recycled polypeptide molded article of the present invention solves the embrittlement problem that plagued conventional polypeptide solidification molded articles, and provides a recycled polypeptide molded article that can be used for practical purposes, such as a medical material.

[0096] 2-2. Structure The regenerated polypeptide molded article of the present invention is an α-βs coexisting polypeptide coagulation molded article containing one or more α-helices in the peptide chain. Conventional polypeptide coagulation molded articles treated with alcohol outside the temperature range of the present invention lose α-helices in the peptide chain due to alcohol treatment, while crystallization of the β-sheet structure is induced, resulting in a βs polypeptide insolubilized molded article with a water-insoluble structure. However, the excessive β-sheet structure resulting from the insolubilization treatment has the problem of changing the physical properties of βs polypeptide insolubilized molded articles to hard and brittle.

[0097] The regenerated polypeptide molded article of the present invention solves the aforementioned problem by being a coagulated polypeptide molded article containing one or more α-helices in the peptide chain, in addition to a β-sheet structure, thereby providing a regenerated polypeptide molded article with a structure that is not brittle and has high ductility.

[0098] The method for producing the recycled polypeptide molded article is not limited, but for example, the method described in the first embodiment is one such method. In this method, an aqueous solution of α-βt coexisting polypeptide is subjected to a coagulation treatment by rapid dehydration using a low-temperature coagulation solution with dehydrating properties. The produced recycled polypeptide molded article contains one or more α-helices in the peptide chain along with the β-sheet structure.

[0099] Specific examples of the recycled polypeptide molded articles of the present invention include thin film molded articles, fine particle molded articles, or fiber molded articles. Furthermore, these recycled polypeptide molded articles may be used as a material to obtain three-dimensional molded articles having a three-dimensional shape. Examples include molded articles used as medical materials (such as bioabsorbable sutures, artificial blood vessels, artificial skin, bioabsorbable scaffolds, and stents).

[0100] The type of polypeptide constituting the regenerated polypeptide molded article of the present invention is not limited, but fibrous proteins such as α-βt / s-coexisting FibH, as described in Example 1, are preferred. Among these, bagworm FibH is particularly preferred.

[0101] <Example 1: Structural confirmation of natural bagworm FibH in aqueous solution> (Objective) To confirm that natural bagworm FibH, which is α-βt / s coexisting FibH, exists in aqueous solution as α-βt coexisting FibH, in which an α-helix and a β-turn coexist within the molecule.

[0102] (Methods) Structural analysis of natural bagworm fibH in aqueous solution was performed by fixing natural bagworm fibH by freeze-drying.

[0103] (1) Preparation of Natural Bagworm FibH Aqueous Solution For the bagworm silk, we used silk spun by the bagworm of the giant bagworm moth. The collected bagworm silk was treated with an aqueous sodium carbonate solution to refine it and obtain natural bagworm FibH. Subsequently, it was dissolved in an aqueous lithium thiocyanate solution and then desalted in a cellulose dialysis tube to obtain an aqueous solution of natural bagworm FibH. The prepared aqueous solution of natural bagworm FibH was calculated to be 1.2% by dry weight measurement.

[0104] Furthermore, a 4.0% aqueous solution of natural bagworm silk was prepared by placing the dialysis-treated FibH aqueous solution into a new cellulose dialysis tube and concentrating it by air drying.

[0105] (2) Preparation of natural bagworm FibH solidified molded product The natural bagworm FibH aqueous solution obtained in (1) was freeze-dried at -80°C using a freeze dryer (EYELA Co., Ltd.) to obtain a regenerated bagworm FibH solidified molded product.

[0106] (3) Wide-angle X-ray diffraction (WAXD) measurement The regenerated bagworm FibH solidified molded product obtained in (2) above was subjected to WAXD measurement. For the measurement, CuKα rays (40kV, 30mA; wavelength 1.5418Å) were measured using a wide-angle X-ray diffractometer T-WAXS (Rigaku Corporation) with a camera length of 50mm, and the measurement results were obtained as a two-dimensional pattern. A hybrid multi-dimensional pixel detector HyPix-6000 (Rigaku Corporation) was used as the detector. A silicon (Si) 111 reflector was used for camera length calibration.

[0107] A 2θ profile was extracted from the obtained two-dimensional pattern, and the 2θ value was obtained for each observed peak. The lattice plane spacing corresponding to each peak was calculated based on Bragg's equation (2d sinθ = λ; d: plane spacing, λ: X-ray wavelength). A value of 1.5418 Å was used for λ.

[0108] (Results) Figure 1 shows the results. In the aqueous solution of natural bagworm FibH, α 10-10 Surface reflection (arrow) and Silk-I 101 Surface reflection (arrowhead) was observed. Silk-I 101 This is a water-soluble crystalline structure composed of aggregates of β-turns. Therefore, it was revealed that natural bagworm FibH exists in aqueous solution as α-βt / s coexisting FibH, in which α-helices and β-turns coexist within the molecule.

[0109] <Example 2: Influence of Coagulation Solution Temperature on the Structure of Regenerated Polypeptide Molded Articles> (Objective) To compare and verify the influence of temperature on the peptide structure of regenerated polypeptide molded articles obtained by dehydration treatment in coagulation solutions at different temperatures using wide-angle X-ray diffraction.

[0110] (Method) Natural bagworm FibH aqueous solution, which is an α-βt coexisting FibH aqueous solution, was rapidly dehydrated in solidification solutions at various temperatures to produce regenerated bagworm FibH solidified molded products, which were then measured by wide-angle X-ray diffraction.

[0111] (1) Preparation of natural bagworm FibH aqueous solution This was carried out in the same manner as in "(1) Preparation of natural bagworm FibH aqueous solution" of Example 1.

[0112] (2) Preparation of Regenerated Bagworm FibH Coated Molded Products The natural bagworm FibH aqueous solution obtained in (1) was extruded into a cooling solidification tank filled with solidification solution to obtain unstretched regenerated bagworm FibH coagulated molded products. 70% ethanol was used as the solidification solution, and the solidification solution temperature was set at five points: 2.5°C, 7°C, 8°C, and 22°C.

[0113] (3) Wide-angle X-ray diffraction (WAXD) measurement The solidified molded products obtained in (2) above, each with different solidification temperatures, were subjected to WAXD measurement. The samples were measured using a wide-angle X-ray diffractometer T-WAXS (Rigaku Corporation) with CuKα rays (40kV, 30mA; wavelength 1.5418Å) at a camera length of 50mm, and the measurement results were obtained as a two-dimensional pattern. A hybrid multi-dimensional pixel detector HyPix-6000 (Rigaku Corporation) was used as the detector. A silicon (Si) 111 reflector was used for camera length calibration.

[0114] For the obtained two-dimensional pattern, 2θ profiles were extracted across all azimuthal angles, and 2θ values ​​were obtained for each observed peak. The lattice plane spacing corresponding to each peak was calculated based on Bragg's equation (2d sinθ = λ; d: plane spacing, λ: X-ray wavelength). A value of 1.5418 Å was used for λ.

[0115] (Results) Figure 2 shows the results. The regenerated bagworm FibH solidified molded products obtained by solidification with solidification solutions at 8°C and 22°C showed the same β as the regenerated bagworm FibH solidified molded products obtained by rapid dehydration treatment with alcohol. 200 As the β-sheet structure of surface reflection (arrowhead) is induced, α 10-10 The surface reflection (arrow) was almost completely absent, revealing that the α-helix had been lost.

[0116] On the other hand, in the regenerated bagworm FibH solidified molded products obtained by solidification in low-temperature solidification solutions at 2.5°C and 7°C, despite rapid dehydration treatment with alcohol, a clear α was observed. 10-10 Surface reflection was observed, indicating the presence of an α-helix.

[0117] <Example 3: Relationship between viscosity and temperature of aqueous solution of α-βt-coexisting polypeptide> (Objective) In Example 2, when a regenerated bagworm FibH solidified molded product was prepared from an aqueous solution of natural bagworm FibH (aqueous solution of α-βt-coexisting polypeptide) in a solidified solution cooled to 7°C or below, a regenerated polypeptide with α-helices remaining in the peptide chain was obtained despite rapid dehydration treatment with alcohol. However, in a solidified solution above 8°C, an insoluble regenerated polypeptide without α-helices was obtained. Therefore, the boundary temperature of the solidified solution that can determine the presence or absence of α-helices after rapid dehydration treatment was investigated from the relationship between the viscosity and temperature of the aqueous solution of α-βt-coexisting polypeptide.

[0118] (Method) (1) Preparation of natural bagworm FibH aqueous solution The same method as in "(1) Preparation of natural bagworm FibH aqueous solution" in Example 1 was used.

[0119] (2) Measurement of the temperature dependence of the viscosity of the natural bagworm FibH aqueous solution The viscosity of the obtained 7.0% natural bagworm FibH aqueous solution was measured at 1°C intervals over a temperature range from 3°C to around 60°C, where gelation begins, and the temperature dependence of the viscosity was evaluated. A laboratory vibrating viscometer VM-10A (Sekonic Corporation) was used for viscosity measurement. Temperature control was performed using a digital hot plate stirrer (model: PC-420D; Corning Corporation), and the heating was set to a solution temperature rise rate of 1°C / min. Solution temperature was measured at each measurement point using a digital controller TTM-004W (model: TTM-004W-PA; Toho Electronics Co., Ltd.).

[0120] (3) For the experiment to measure the temperature dependence of viscosity in an aqueous solution of mutant bagworm FibH, a 7.0% aqueous solution of C-terminally deficient mutant bagworm FibH was used. Viscosity was measured at 0.5°C intervals over a temperature range from around 1.3°C to around 60°C, where gelation begins, and the temperature dependence of viscosity was evaluated. A laboratory vibrating viscometer VM-10A (Sekonic Corporation) was used for viscosity measurement. Temperature control was performed using a digital hot plate stirrer (Corning Inc.; part number: PC-420D), and heating was performed so that the solution temperature rose at a rate of approximately 1°C / min. Solution temperature was measured at each measurement point using a digital controller TTM-004W (model: TTM-004W-PA; Toho Electronics Co., Ltd.).

[0121] (Results) The results are shown in Figures 3 and 4. These figures plot the viscosity of the natural bagworm FibH aqueous solution (Figure 3) or the mutant bagworm FibH aqueous solution (Figure 4) at each temperature. The curvature of the correlation curve between temperature and viscosity plots remained constant at 6-7°C in Figure 3 and at 3-7°C in Figure 4, both of which were consistent with the change at the boundary temperature of 7-8°C shown in Example 2. Therefore, the results from Examples 2 and 3 indicate that 6-7°C is the inflection temperature at which the viscosity change inflection point occurs.

[0122] From the above results, it became clear that when the coagulation solution is a lower alcohol, regenerated polypeptides can be obtained that retain their α-helix structure even after rapid dehydration with alcohol, as long as the temperature is below the inflection temperature of 6°C to 7°C.

[0123] <Example 4: Verification of the stretchability of the mutant bagworm FibH solidified molded product> (Objective) To compare and verify the tensile strength (stretchability) of each molded product when subjected to stretching, and the regenerated bagworm FibH solidified molded product obtained by solidification treatment in alcohol solidification solutions at different temperatures.

[0124] (Methods) (1) Preparation of mutant bagworm FibH aqueous solution A mutant bagworm FibH was constructed and an aqueous solution thereof was prepared. The recombinant bagworm FibH gene was created by cloning the bw753 gene according to the method disclosed in Example 1 of WO2020 / 235692. The "bw753 gene" (bagworm-753aa coding gene) consists of the nucleotide sequence shown in Sequence ID No. 15 and encodes the bw753 protein, which is a recombinant bagworm FibH derived from the giant bagworm moth. The "bw753 protein" consists of 753 amino acids shown in Sequence ID No. 14 and is an α-βt / s coexisting FibH composed of an N-terminal region, a central region containing four repeat units, and a C-terminal region.

[0125] Furthermore, a bw753 gene expression vector for expressing the bw753 gene in E. coli was constructed using the expression vector pET-26b(+) (Novagen) in accordance with the method disclosed in Example 2 of WO2020 / 235692. The resulting expression vector was named "pET-26b-bw753".

[0126] After purifying the constructed expression vector (pET-26b-bw753), it was introduced into E. coli BLR (DE3) strain cells (Novagen) using a standard method to prepare E. coli transformants. Induction of protein expression in the E. coli transformants and extraction and purification of the solubilized bw753 protein were performed according to the methods disclosed in Examples 1 and 4 of WO2024 / 057361. Subsequently, the resulting mutant bagworm FibH aqueous solution was air-dried and concentrated to prepare a 6% mutant bagworm FibH aqueous solution. This aqueous solution was used in the subsequent steps.

[0127] (2) Preparation of Regenerated Bagworm FibH Coated Molded Products The mutant bagworm FibH aqueous solution obtained in (1) was extruded in a cooling solidification tank filled with solidification solution to obtain unstretched regenerated bagworm FibH coated molded products. 70% ethanol was used as the solidification solution, and the solidification solution temperatures were set at three points: 5°C, 12°C, and 15°C. Of these, only the solidification solution temperature below the inflection temperature was 5°C.

[0128] (3) Tensile Test Tensile tests were performed on solidified molded products of unstretched regenerated bagworm FibH, molded at temperatures of 5°C and 15°C with an initial length of 12.5 mm and a tensile speed of 5 mm / min in an environment of 25°C and 52 RH%. The cross-section of the solidified molded product, cut perpendicular to the length direction with a razor blade, was observed using a scanning electron microscope (KEYENCE; model: VHX-D510) to measure the initial cross-sectional area of ​​the test sample. The relationship between force (vertical axis) and displacement (horizontal axis) obtained from the tensile test was divided by the initial cross-sectional area of ​​the test sample and the initial length of the sample (length between grips), respectively, to obtain the relationship between stress (vertical axis) and strain (horizontal axis). The elastic modulus (GPa) is given by the initial slope of the obtained stress-strain curve, the fracture strength (MPa) is the stress value just before fracture, the fracture elongation (fracture strain) (%) is the strain at fracture, and the toughness (MJ / m²) is given by the total integral area of ​​the stress-strain curve. 3 ) was estimated.

[0129] (4) Heat stretching treatment Each unstretched regenerated bagworm FibH solidified molded product obtained in (2) was immersed in an oil bath heated to 140°C and heat stretched at three different stretching ratios (3.79 times, 3.45 times, and 2.92 times), and the stretching duration until the solidified molded product broke was verified. Heat stretching was performed 1 to 3 times for each unstretched solidified molded product. If stretching continued for 60 seconds, it was forcibly terminated, and the centrifugal stretching duration was recorded as 60 seconds. In addition, if no stretching was possible and the product broke immediately, the stretching duration was recorded as 0 seconds.

[0130] (Results) The results of the tensile test are shown in Table 1, and the results of the heat stretching are shown in Tables 2 to 4.

[0131]

[0132] As shown in Table 1, no significant difference was observed in the elastic modulus and fracture strength of the recycled bagworm FibH solidified molded products extruded in solidification solutions at 5°C and 15°C. However, a difference of more than 100 times was observed in fracture elongation (fracture strain) and toughness between the 5°C and 15°C extrusion processes. This result demonstrates that unstretched solidified molded products molded at temperatures below the inflection temperature show significantly suppressed embrittlement, which is observed in recycled polypeptide molded products that have undergone conventional rapid dehydration treatment with alcohol, and that their ductility is improved.

[0133] Tables 2-4 show the results for stretch ratios of 3.79, 3.45, and 2.92, respectively.

[0134]

[0135]

[0136] In the table, (-) indicates that the experiment has not been attempted.

[0137] Furthermore, the results shown in Tables 2 to 4 indicate that, regardless of the draw ratio, the draw duration clearly decreases as the solidification solution temperature increases, making heat draw difficult. On the other hand, in the case of regenerated bagworm FibH extruded with a solidification solution at 5°C, it was shown that the draw duration lasted at least 50 to 60 seconds even under conditions of a draw ratio of 3 times or more (draw strain of 200% or more). These results clearly demonstrate that the regenerated polypeptide solidified molded product of the present invention exhibits suppressed embrittlement in low-temperature solidification solutions and possesses physical properties that allow for high heat draw. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for producing a recycled polypeptide molded article, comprising a coagulation step of coagulating an aqueous solution of an α-βt / s coexisting polypeptide containing one or more α-helix-forming sequences and β-turn-forming sequences in one molecule by contact with a coagulation solution, wherein the α-helix-forming sequence consists of an amino acid sequence that forms an α-helix in whole or in part in the aqueous solution or the polypeptide coagulated molded article, the β-turn-forming sequence consists of an amino acid sequence that forms a β-turn in whole or in part in the aqueous solution and forms a β-sheet structure in whole or in part in the polypeptide coagulated molded article, and the temperature of the coagulation solution in the coagulation step is -3°C or higher and below the inflection temperature in the temperature-viscosity correlation curve.

2. The manufacturing method according to claim 1, comprising a dissolution step of dissolving the α-βt / s coexisting polypeptide in water before the coagulation step to prepare an aqueous solution of the α-βt coexisting polypeptide.

3. The manufacturing method according to claim 1 or 2, further comprising a stretching step of stretching the regenerated polypeptide molded product obtained after the solidification step.

4. The manufacturing method according to any one of claims 1 to 3, wherein the α-helix-forming sequence consists of the amino acid sequence shown in SEQ ID NO: 1 and / or the amino acid sequence shown in SEQ ID NO:

2.

5. The manufacturing method according to any one of claims 1 to 4, wherein the β-turn forming sequence consists of the amino acid sequence shown in SEQ ID NO: 3 and / or the amino acid sequence shown in SEQ ID NO:

4.

6. The method for producing a protein by any one of claims 1 to 5, wherein the α-βt / s coexisting polypeptide comprises an α-βt / s coexisting fibroin H chain protein.

7. The method for producing fibroin H chain protein according to claim 6, wherein the α-βt / s coexisting fibroin H chain protein includes a mutant fibroin H chain protein.

8. The manufacturing method according to claim 7, wherein the α-βt / s coexisting fibroin H chain protein is derived from bagworm silk.

9. The manufacturing method according to any one of claims 1 to 8, wherein the coagulation solution is a solution containing a lower alcohol.

10. The manufacturing method according to claim 9, wherein the concentration of the lower alcohol in the solution containing the lower alcohol is 30% to 90%.

11. The manufacturing method according to claim 10, wherein the inflection temperature is 6°C to 7°C.

12. A molded article of a regenerated polypeptide containing one or more α-helix structures in the molecule.

13. The molded article of the regenerated polypeptide according to claim 12, wherein the regenerated polypeptide comprises fibroin H chain protein.

14. The recycled polypeptide molded article according to claim 13, which is a thin film molded article, a fine particle molded article, or a fiber molded article.

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