Fibroin h-chain aqueous solution for processing polypeptide molded article, polypeptide molded article using same, and method for producing same

Abstract

The objective of the present invention is to develop a regenerated fibroin H-chain protein molded article which does not require an insolubilization treatment, does not involve forming excessive β-sheet structure, is insoluble in water and exhibits malleability, flexibility and moderate strength.　More specifically, provided is a fibroin H-chain aqueous solution for processing a polypeptide molded article, wherein the fibroin H-chain aqueous solution contains a fibroin H-chain protein that includes one or more α-helix formation sequences and β-turn formation sequences in one molecule.

Description

Aqueous solution of fibroin H chain for processing polypeptide molded articles, polypeptide molded articles using the same, and methods for producing the same.

[0001] The present invention relates to an aqueous solution of fibroin H chain for processing polypeptide molded articles, a polypeptide molded article produced using the same, and a method for producing the same.

[0002] Synthetic resins, such as plastics, are used in every aspect of human society and have become indispensable materials for modern life. However, on the other hand, problems such as global warming caused by the large amount of carbon dioxide released when they are burned, and environmental pollution from waste plastics such as microplastics due to their chemical stability and lack of biodegradability, have become apparent.

[0003] Therefore, in recent years, in order to realize a sustainable society, there has been a demand for the development of environmentally friendly and safe alternatives to synthetic resins made from fossil fuels. Polypeptide molded products, for example, are one such alternative.

[0004] Fibroin protein is attracting attention as a raw material for polypeptide molded products. Fibroin protein is a fiber component of silkworm silk and spider silk (dragging silk), and because it has a β-sheet structure as its crystalline structure, it exhibits high toughness with an excellent balance of strength and elongation. Furthermore, because it is composed of polypeptides, it is biodegradable and has high biocompatibility. For this reason, polypeptide molded products made from fibroin protein are expected to be used as materials in a wide range of fields, such as medical materials and structural materials.

[0005] Generally, when processing fibroin protein into polypeptide molded products, liquid protein is used as the raw material. Examples include liquid fibroin protein stored in the silk glands of silkworms (so-called liquid silk) before fiber formation, and liquid fibroin protein obtained by expressing it in E. coli or other bacteria using genetic engineering technology. In addition, an aqueous solution of fibroin protein, obtained by dissolving already fiber-formed solid fibroin protein in a solvent to make it liquid, can also be used as a raw material. Regenerated fibroin protein, obtained by coagulating an aqueous solution of fibroin protein to make it solid again, is expected to have applications 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, insolubilization treatment by alcohol treatment or heat treatment is necessary during the molding process.

[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 insolubilization. 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 eri silkworm (Samia cynthia ricini) and spider fibroin H-chain protein. However, the polypeptide coagulated molded articles obtained by dehydrating these aqueous solutions contain α-helices, and are therefore soluble in water, similar to silkworms. As a result, insolubilization treatment is essential during molding, and the material becomes hard and brittle.

[0009] Kim HH, et al., 2016, Polymer, 90: 26-33.Guo C., et al. 2020, Biomacromolecules, 21: 1678-1686.

[0010] We have developed and provide an aqueous solution of fibroin H-chain protein that does not require insolubilization treatment, does not involve excessive β-sheet structuring, is insoluble in water, and possesses ductility, flexibility, and moderate strength, making it a raw material for molded products of regenerated fibroin H-chain protein.

[0011] To solve the above problems, the inventors conducted extensive research and found that a polypeptide coagulation molded product obtained by dehydrating an aqueous solution of fibroin H-chain protein has α-helix and β-sheet structures in its molecule, is insoluble in water, does not require insolubilization treatment, is highly ductile, flexible, and has moderate strength.

[0012] Furthermore, it has become clear that even fibroin H-chain proteins modified by genetic engineering technology exhibit similar physical properties in polypeptide coagulated products, as long as they possess a specific amino acid sequence. This invention is based on the results of the aforementioned research and development and includes the following:

[0013] (1) A fibroin H chain aqueous solution for processing polypeptide molded articles, comprising a fibroin H chain protein containing one or more α-helix-forming sequences and β-turn-forming sequences in one molecule, 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 in a polypeptide coagulated molded article obtained by dehydrating the aqueous solution, and 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 a polypeptide coagulated molded article obtained by dehydrating the aqueous solution. (2) The aqueous solution according to (1), 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. (3) The aqueous solution according to (1) or (2), 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. (4) An aqueous solution according to any one of (1) to (3), wherein all or part of the fibroin H chain protein is a mutant fibroin H chain protein. (5) An aqueous solution according to any one of (1) to (4), wherein the fibroin H chain protein is derived from bagworm silk. (6) A polypeptide coagulated molded article obtained by dehydrating the aqueous solution according to any one of (1) to (5). (7) A polypeptide molded article obtained by molding the polypeptide coagulated molded article according to (6).

[0014] (8) A method for producing a polypeptide molded article that does not require an insolubilization treatment, comprising a coagulation step of dehydrating and coagulating an aqueous solution of fibrous protein for processing polypeptide molded articles to obtain a polypeptide coagulated molded article, wherein the aqueous solution of fibrous protein is an aqueous solution containing a fibrous protein that each contains one or more α-helix forming sequences and β-turn forming sequences in one molecule, 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 in the polypeptide coagulated molded article obtained by dehydrating the aqueous solution, and the β-turn forming sequence consists of an amino acid sequence that forms a β-turn in whole or in part in the polypeptide coagulated molded article obtained by dehydrating the aqueous solution, and forms a β-sheet structure in whole or in part. (9) The method for producing a polypeptide molded article according to (8), comprising a molding step of molding the polypeptide coagulated molded article into a desired shape. (10) The method for producing a polypeptide molded article according to (8) or (9), 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 GGX (where X is A or S) or SEQ ID NO: 2. (11) The method for producing according to any one of (8) to (10), wherein the β-turn forming sequence consists of GXGXGX (where X is A or S), or the amino acid sequence shown in SEQ ID NO: 3 and / or the amino acid sequence shown in SEQ ID NO: 4. (12) The method for producing according to any one of (8) to (11), wherein the fibrous protein is a fibroin H chain protein.

[0015] According to the aqueous fibroin H-chain solution for polypeptide molded articles of the present invention, by using the aqueous solution as a raw material for molding, a water-insoluble molded article can be obtained without the need for insolubilization treatment.

[0016] This figure compares the wide-angle X-ray diffraction results of regenerated bagworm FibH cast films obtained by molding from a natural bagworm FibH aqueous solution, both before and after immobilization treatment. A shows the regenerated bagworm FibH cast film, which is a polypeptide coagulation molded product of the present invention, without immobilization treatment, and B shows the regenerated bagworm FibH cast film after heat treatment as an immobilization treatment, which is a polypeptide immobilization molded product. This figure shows the wide-angle X-ray diffraction results of the regenerated mutant bagworm FibH cast film, which is a polypeptide coagulation molded product of the present invention, obtained by molding from a mutant bagworm FibH aqueous solution. This figure shows the wide-angle X-ray diffraction results of the polypeptide coagulation molded product of the present invention, obtained by freeze-drying a natural bagworm FibH aqueous solution. This figure compares the wide-angle X-ray diffraction results of regenerated FibH cast films, which are polypeptide coagulation molded products of the present invention, obtained by molding from natural FibH aqueous solutions derived from various organisms, without immobilization treatment. The results for regenerated FibH cast films are shown for silkworms (Bombyx mori), Eri silkworms (Samia cynthia ricini), and the giant bagworm (Eumeta japonica). The figure shows the results of tensile tests on regenerated FibH cast films of immobilized silkworms (a) and unimmobilized bagworms (b).

[0017] 1. Aqueous solution of fibroin H chain for polypeptide molded articles 1-1. Overview The first aspect of the present invention is an aqueous solution of fibroin H chain for polypeptide molded articles. The present invention relates to an aqueous solution in which fibroin H chain protein is dissolved as a raw material for polypeptide molded articles. The fibroin H chain protein is characterized in that, in the aqueous solution, it forms one or more α-helices and β-turns in one molecule, and in the coagulated product obtained by dehydration treatment, it forms one or more α-helices and β-sheet structures in one molecule.

[0018] According to the aqueous fibroin H chain aqueous solution for polypeptide molded articles of the present invention, by coagulating it, a polypeptide coagulated molded article that is insoluble in water, highly ductile, flexible, and has moderate strength can be obtained without insolubilization treatment. Furthermore, by adjusting the ratio of α-helix-forming sequences and β-turn-forming sequences in the amino acid sequence of the fibroin H chain protein, it is possible to control the physical properties of the regenerated polypeptide molded article.

[0019] 1-2. Definitions The following terms used herein are defined below.

[0020] 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.

[0021] 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.

[0022] 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 mainly refers to regenerated fibroin H-chain protein obtained from an aqueous solution of fibroin H-chain protein.

[0023] In this specification, "recycled polypeptide molded article" refers to a polypeptide coagulation molded article or a polypeptide insolubilized molded article obtained by molding a recycled polypeptide to have a certain hardness and a specific shape. Examples include thin film molded articles, fine particle molded articles, or fiber molded articles.

[0024] In this specification, "polypeptide-coagulated molded article" means a regenerated polypeptide molded article obtained by coagulating an aqueous polypeptide solution, for example, by dehydration, and without insolubilization 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 polypeptide-coagulated molded article).

[0025] In this specification, "polypeptide-insolubilized molded article" refers to a regenerated polypeptide molded article that has been immobilized, obtained by dehydrating and coagulating an aqueous polypeptide solution. Polypeptide-insolubilized molded articles typically refer to β-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. In this specification, polypeptide molded articles obtained by immobilizing a polypeptide coagulated molded article also fall under the category of polypeptide-insolubilized molded articles.

[0026] In this specification, "solidification" means the process by which a liquid becomes a solid, i.e., solidification.

[0027] In this specification, "immobilization treatment" refers to a treatment that immobilizes a regenerated polypeptide. Specific examples of immobilization treatments, though not limited to these, include heat treatment, alcohol treatment, stretching treatment, or combinations thereof.

[0028] 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 organism of origin. When referring to silk thread derived from a specific organism, the name of that organism shall be placed before "silk thread," such as silkworm silk or bagworm silk.

[0029] 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.

[0030] 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.

[0031] 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 has a β-sheet structure as the main fiber crystal structure of natural silk thread.

[0032] 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.

[0033] 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 obtained by artificially modifying all or part of natural FibH through genetic recombination technology or the like.

[0034] 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 above-mentioned 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.

[0035] As used herein, the term "regenerated fibroin heavy chain protein" (regenerated FibH) refers to a product obtained by dissolving FibH in a solvent to turn it into a liquid state, and then the FibH coagulates to become a solid state having a specific shape again.

[0036] 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 it contains 3.6 amino acid residues per turn.

[0037] 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.

[0038] As used herein, "β-sheet structure" is one of the secondary structures in a polypeptide, in which at least 2 to 3 β-chains (β-strands) adjacent in parallel are laterally linked by intermolecular hydrogen bonds to form a pleated sheet structure that is twisted as a whole. A substance having a β-sheet structure in the molecule may be insoluble in water.

[0039] 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.

[0040] 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.

[0041] As used herein, the term "aqueous solution" refers to a solution that uses water as the main solvent among liquid mixtures composed of two or more substances. The solute dissolved in the aqueous solution may be solid, liquid or gas. Examples of solutes include electrolytes and water-soluble substances. In addition, liquid solutes may also include organic solvents such as lower alcohols.

[0042] 1-3. Composition The FibH aqueous solution for molded polypeptide articles of the present invention is composed of an aqueous solution in which α-βt / s coexisting FibH is dissolved.

[0043] As used herein, "α-βt / s coexisting FibH" (α-helix-β-turn / sheet coexisting fibroin H chain protein) is a fibroin H chain protein characterized by comprising one or more α-helix forming sequences and one or more β-turn forming sequences respectively in a single FibH molecule. Due to this characteristic, α-βt / s coexisting FibH has the property of allowing α-helices and β-turns 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 polypeptide article.

[0044] In this specification, "α-βt coexisting FibH aqueous solution" (α-helix-β-turn coexisting FibH aqueous solution) is an aqueous solution in which α-βt / s coexisting FibH is dissolved, and is synonymous with the FibH aqueous solution for polypeptide molded articles of the present invention. As described above, α-βt / s coexisting FibH has α-helices and β-turns coexisting in a stable molecular form within the peptide chain in the aqueous solution. The α-βt / s coexisting FibH contained in the α-βt coexisting FibH aqueous solution may be of the same type or of different types.

[0045] 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 solution or a polypeptide coagulated molded article. For example, an amino acid sequence represented by (A)n (where n is an integer ≥ 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 is an integer ≥ 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 longer than that of an α-helix, but in this specification, the "31-helix" is also included as a structure of the α-helix.

[0046] 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 solution, 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 an 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 (S), and n is an integer ≥ 3), specifically the amino acid sequence shown in Sequence ID No. 3. Also, 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.

[0047] α-βt / s coexisting FibH is defined as having 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 α-βt / s coexisting FibH, the α-helix-forming sequences and β-turn-forming sequences may form separate clusters in multiple consecutive sequences, or they may alternate consecutively. Furthermore, the α-helix-forming sequences and β-turn-forming sequences may be randomly distributed within the amino acid sequence of α-βt / s coexisting FibH.

[0048] In α-βt / s coexisting FibH, the FibH can be either natural or artificial.

[0049] An example of a natural fibH having the α-βt / s coexisting fibH structure is the fibroin H chain protein 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 α-βt / s coexisting fibH, bagworm fibH is one of the few natural fibHs that possesses the characteristics of α-βt / s coexisting fibH.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 the repeating unit, such as the N-terminal region and / or the C-terminal region.

[0056] An example of artificial FibH having a structure in which α-βt / s coexist is mutant bagworm FibH.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] Specific examples of mutant bagworm FibH 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.

[0061] 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.

[0062] Another example of a mutant bagworm FibH is the terminal mutant bagworm FibH, which contains mutations in either the N-terminal region, the C-terminal region, or both of the wild-type bagworm FibH.

[0063] 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.

[0064] 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.

[0065] Specific examples of terminal region 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 terminal region mutant bagworm FibHΔC, which is a mutant bagworm FibH having 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 terminal region mutant bagworm FibHΔN, which is a mutant bagworm FibH having 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 terminal region mutant bagworm FibHΔN / C, which is a mutant bagworm FibH having the amino acid sequence shown in SEQ ID NO: 14, but with both the N-terminal and C-terminal regions completely deleted.

[0066] Examples of specific base sequences of genes encoding terminal region mutant FibH include the terminal region mutant bagworm FibHΔC gene shown in SEQ ID NO: 19, the terminal region mutant bagworm FibHΔN gene shown in SEQ ID NO: 20, and the terminal region mutant bagworm FibHΔN / C gene shown in SEQ ID NO: 21.

[0067] 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.

[0068] 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 techniques. 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.

[0069] 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).

[0070] 2. Polypeptide Coagulated Molded Article 2-1. Overview The second aspect of the present invention is a polypeptide coagulated molded article. This is a polypeptide molded article obtained by coagulating the FibH aqueous solution for polypeptide molded articles described in the first aspect of the present invention. According to the polypeptide coagulated molded article of the present invention, it is possible to provide a highly sustainable α-βs-coagulated FibH coagulated molded article that is insoluble in water, has high ductility, is flexible and has moderate strength. Furthermore, a polypeptide molded article having similar physical properties can be obtained by molding it.

[0071] 2-2. Structure The polypeptide coagulated molded article of the present invention is an α-βs-coagulated FibH molded article obtained by coagulating α-βt / s-coagulated FibH dissolved in the aqueous solution of FibH for polypeptide molded articles described in the first embodiment.

[0072] In this specification, "α-βs coexisting FibH coagulated molded article" (α-helix-β-sheet coexisting FibH coagulated molded article) refers to a polypeptide coagulated molded article obtained by coagulating the aqueous solution of α-βt coexisting FibH of the present invention, which is an aqueous solution of α-βt / s coexisting FibH. As described above, due to the properties of α-βt / s coexisting FibH, the α-helix and β-sheet structures coexist in the peptide chain as metastable molecular forms in the polypeptide coagulated molded article.

[0073] In the polypeptide solidified molded article of the present invention, the solvent, water, may be completely removed, or some may remain. The water content of the polypeptide solidified molded article is not limited, but can be 20%db or less, 15%db or less, 10%db or less, 8%db or less, 6%db or less, 5%db or less, 4%db or less, 3%db or less, 2%db or less, 1%db or less, 0.5%db or less, or 0.1%db or less on a dry-base basis (dB). The lower limit is 0%db. Generally, a higher water content is preferable when the polypeptide solidified molded article is flexible, and a lower water content is preferable when a predetermined shape and hardness are required.

[0074] The polypeptide coagulated molded article of the present invention may contain other components besides α-βt / s coexisting FibH. For example, in addition to the residual solvent (water) mentioned above, it may also contain non-α-βt / s coexisting FibH such as silkworm FibH or spider FibH, low molecular weight compounds (including dyes, monosaccharides, oligosaccharides, drugs and pharmaceutical compositions), other polypeptides other than FibH (including antibodies), nucleic acids, salts, polymers (including polysaccharides, PEG, etc.), and the like.

[0075] The polypeptide solidified molded product of the present invention may be in an unmolded state or a molded polypeptide molded product.

[0076] α-βs coexisting FibH coagulated molded products, which are polypeptide coagulated molded products, have FibH, a fiber component of silk yarn with a β-sheet structure in its crystalline structure, as their main component. Therefore, they exhibit high toughness with an excellent balance of strength and elongation inherent in FibH. Furthermore, because their main component is polypeptide, they possess high biocompatibility and natural decomposition. In addition, despite being a coagulated molded product, they are insoluble in water, thus lacking the brittleness of conventional recycled FibH that undergoes insolubilization treatment. They possess high ductility, flexibility, and moderate strength.

[0077] The types of polypeptide-coagulated molded articles of the present invention are not limited. They encompass molded articles of various forms obtained by molding processes. Specifically, examples include molded articles as materials such as fibers, films (including sheets), or plates, as well as medical materials (artificial fibers, regenerative medicine materials) or structural materials based on such materials.

[0078] 3. Method for producing polypeptide molded articles without immobilization treatment 3-1. Overview The third aspect of the present invention is a method for producing polypeptide molded articles without immobilization treatment. The production method of the present invention includes a coagulation step and a molding step, and uses an aqueous solution of α-βt-coexisting fibrous protein as a raw material to produce a molded article having α-βt / s-coexisting fibrous protein as the main component. According to the production method of the present invention, a polypeptide molded article that is insoluble in water can be produced without the need for immobilization treatment.

[0079] 3-2. Method The manufacturing method of the present invention includes a solidification step and a molding step. Of these, the solidification step is an essential step, and the molding step is an optional step. Each step will be described in detail below.

[0080] (1) Coagulation process The "coagulation process" is a process in which an aqueous solution of fibrous protein containing α-βt is dehydrated and coagulated to obtain a molded product of fibrous protein containing α-βs.

[0081] In this specification, "α-βt / s coexisting fibrous protein" refers to a fibrous protein characterized by containing one or more α-helix-forming sequences and β-turn-forming sequences within a single fibrous protein molecule. If the fibrous protein is FibH, it becomes the α-βt / s coexisting FibH described in the first embodiment.

[0082] In this specification, "α-βt coexisting fibrous protein aqueous solution" refers to an aqueous solution obtained by dissolving an α-βt / s coexisting fibrous protein in water, in which the α-helix and β-turn coexist in a stable molecular form within the peptide chain. The α-βt coexisting FibH aqueous solution described in the first embodiment is one form of this α-βt coexisting fibrous protein aqueous solution, and corresponds to the case where the fibrous protein is FibH.

[0083] In this specification, "α-βs coexisting fibrous protein coagulated molded product" refers to a polypeptide coagulated molded product obtained by dehydrating and coagulating an aqueous solution of α-βt coexisting fibrous protein, wherein the α-helix and β-sheet structures coexist in a metastable molecular form within the peptide chain. The α-βs coexisting FibH coagulated molded product described in the first embodiment is one form of this α-βs coexisting fibrous protein coagulated molded product, and corresponds to the case where the fibrous protein is FibH.

[0084] In this specification, "dehydration treatment" refers to a process of removing the solvent, i.e., water, from an aqueous solution of α-βt-coexisting fibrous protein in order to coagulate the α-βt / s coexisting fibrous protein. The specific method of dehydration treatment is not limited as long as it can make the α-βs-coexisting fibrous protein visible as a solid. Examples include drying and dehydration using a coagulation solution.

[0085] In this specification, "drying" means reducing moisture content by evaporation or sublimation.

[0086] The drying method is not limited as long as it can reduce the moisture content in the α-βt coexisting fibrous protein aqueous solution. Examples include natural drying by exposure to the open air, dehumidification drying by sealing the solution in a sealed container with a desiccant, sun drying, ventilation drying by blowing hot air, heat drying, far-infrared drying, low-temperature drying, vacuum drying by degassing, freeze-drying, or a combination thereof. All of these techniques are well known and should be carried out according to conventional methods.

[0087] The drying time varies depending on the drying method, temperature, humidity, and the desired residual water content of the polypeptide solidified molded product after the desolvent treatment, so it should be determined appropriately according to each condition.

[0088] "Dehydration by coagulation solution" refers to the process of extruding a liquid polymer (in this specification, an aqueous solution of α-βt-containing fibrous protein) from a nozzle into a liquid tank filled with a coagulation solution, thereby reducing the water content and causing coagulation. This method often involves molding simultaneously with coagulation by dehydration, for example, the wet fiber molding method and wet film formation method described in "(2) Molding Process" below.

[0089] Furthermore, water may be completely removed or partially removed. "Partial removal" means reducing the amount of water in the aqueous solution.

[0090] A polypeptide solidified molded product can be obtained after this process.

[0091] (2) Molding Process The "molding process" is the process of molding the polypeptide solidified molded product into the desired shape. In the manufacturing method of the present invention, this is an optional process and may be performed as needed. This process can be performed after the solidification process or simultaneously with the solidification process. It is not necessary to perform this process immediately after the solidification process; for example, this process may be performed when necessary after the polypeptide solidified molded product has been stored or transported in that state.

[0092] The processing technology for molding polypeptide solidified molded articles should follow known methods tailored to the target molded article. The types of molded articles are not limited, but as mentioned above, examples include fibers, films (including sheets), or plates. The molding method for each type of molded article should apply known technologies from the manufacturing fields of synthetic resins, chemical fibers, etc., and the specific method should be carried out according to the respective molding method.

[0093] One example of a method for forming a fibrous shape is a fiber molding method. The "fiber molding method" is a method of drawing a liquid polymer (in this specification, an aqueous solution of α-βt coexisting fibrous protein) from a nozzle, solidifying it, and forming it into fibers. Therefore, in this method, the solidification step and the molding step are performed simultaneously.

[0094] Known fiber molding methods include dry fiber molding, wet fiber molding, molten fiber molding, emulsion fiber molding, and gel fiber molding. While not limited to these methods, dry fiber molding or wet fiber molding is preferred in this process.

[0095] The "dry fiber forming method" is a method of forming fibers by extruding a solution containing a dissolved polymer (in this specification, an aqueous solution of α-βt-containing fibrous protein) from a die into the air and evaporating the solvent. The "wet fiber forming method" is a method of forming fibers by extruding a solution containing a polymer dissolved in a solvent (in this specification, an aqueous solution of α-βt-containing fibrous protein) into a suitable coagulation solution.

[0096] Methods for forming the film include, for example, solution casting, wet film deposition, and extrusion molding. While not limited to these methods, solution casting or wet film deposition is preferred in this process.

[0097] The "solution casting method" is a method of forming a film by pouring a solution (dope; in this specification, an aqueous solution of α-βt coexisting fibrous protein) in which a polymer has been dissolved in a solvent to give it fluidity onto a drum with a smooth surface (casting drum) or a smooth stainless steel belt, allowing it to adhere to the surface, and then heating the drum or smooth belt to evaporate the solvent.

[0098] "Wet film formation method" is a method of forming a film by extruding a solution in which a polymer is dissolved (in this specification, an aqueous solution of α-βt coexisting fibrous protein) into a suitable coagulation solution.

[0099] Methods for forming the material into plates include solution casting and injection molding.

[0100] The molded product obtained using the above molding method can be further subjected to secondary molding processes. For example, a sheet-like molded product can be molded into a three-dimensional shape using methods such as vacuum forming or press forming.

[0101] <Example 1: Structural comparison of natural bagworm FibH molded products with and without insolubilization treatment> (Objective) To compare and verify the peptide structure of regenerated polypeptide molded products of natural bagworm FibH, which is α-βt / s coexisting FibH, with and without conventional insolubilization treatment, i.e., in the case of polypeptide coagulation molded products, using wide-angle X-ray diffraction.

[0102] (Method) (1) Preparation of natural bagworm FibH aqueous solution For the bagworm silk, we used silk spun by the bagworm of the giant bagworm moth.

[0103] The harvested bagworm silk threads were treated with a sodium carbonate aqueous solution to refine them and obtain natural bagworm fibH. Subsequently, they were dissolved in a lithium thiocyanate aqueous 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 cast onto a plastic petri dish and dried for 14 days under wind-free conditions in a constant temperature and humidity chamber at 20°C and 90% relative humidity to produce a recycled bagworm FibH cast film with a thickness of approximately 0.5 mm as an α-βs coexisting FibH solidified molded product.

[0106] (3) Heat treatment of regenerated bagworm FibH cast film The regenerated bagworm FibH cast film obtained in (2) was cut in half. One half was left as an untreated regenerated bagworm FibH cast film (α-βs coexisting FibH solidified molded product), and the other half was placed between two glass slides and heat-treated on a hot plate at 196 (±2) °C for 5 minutes to insolubilize it. After that, it was rapidly cooled at room temperature to produce a regenerated bagworm FibH cast film after insolubilization (heat treatment).

[0107] (4) Wide-angle X-ray diffraction (WAXD) measurement Test pieces measuring 5 mm × 1 mm were cut from each of the two types of regenerated bagworm FibH cast films obtained in (3) above and subjected to WAXD measurement. For the measurement, CuKα rays (40 kV, 30 mA; wavelength 1.5418 Å) were measured using a wide-angle X-ray diffractometer T-WAXS (Rigaku Corporation) with a camera length of 50 mm, 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.

[0108] 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.

[0109] (Results) Figure 1 shows the results. In the regenerated bagworm FibH cast film A, which was not treated for insolubilization, α10-10 Surface reflection and β 200 / 210 Surface reflection and β 211 The coexistence of surface reflections was confirmed. From this result, it was proven that the regenerated bagworm FibH cast film, which is natural bagworm FibH with coexisting α-βt / s structures and has not undergone insolubilization treatment, contains both α-helices and β-sheet structures in its molecule. On the other hand, the regenerated bagworm FibH cast film after heat treatment as an insolubilization treatment showed the presence of α-helices, similar to conventional regenerated FibH molded products such as silkworm silk and spider silk. 10-10 Surface reflection almost disappears, β 200 / 210 Surface reflection and β 211 A significant improvement in surface reflection intensity was observed.

[0110] <Example 2: Structural confirmation of a solidified mutant bagworm FibH product> (Objective) For artificially constructed mutant bagworm FibH, if it is α-βt / s coexisting FibH, the solidified product of the mutant bagworm FibH obtained by solidifying its aqueous solution and without insolubilization treatment will have α-helices and β-sheet structures in its molecule, similar to a solidified product of natural bagworm FibH.

[0111] (Method) (1) Preparation of mutant bagworm FibH The mutant 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 mutant bagworm FibH derived from the giant bagworm moth. The "bw753 protein" consists of 753 amino acids shown in Sequence ID No. 14 and is composed of an N-terminal region, a central region containing four repeat units, and a C-terminal region.

[0112] Furthermore, the 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".

[0113] After purifying the constructed expression vector (pET-26b-bw753), E. coli BLR (DE3) strain (Novagen) was introduced into cells using a standard method to prepare E. coli transformants. Protein expression induction and extraction and purification of the solubilized bw753 protein in the E. coli transformants were performed according to the methods disclosed in Examples 1 and 4 of WO2024 / 05361.

[0114] For expression confirmation after purification, the bw753 protein was subjected to electrophoresis on a 10% SDS-polyacrylamide gel according to the method disclosed in Example 4 of WO2024 / 05361, followed by total protein staining with CBB stain to confirm its expression.

[0115] (2) Preparation of mutant bagworm FibH aqueous solution The mutant bagworm FibH aqueous solution prepared in (1) was air-dried and concentrated to prepare a 2% mutant bagworm FibH aqueous solution. This aqueous solution was used in the subsequent steps.

[0116] (3) Preparation of mutant bagworm FibH solidified molded product A regenerated mutant bagworm FibH cast film was obtained by following the method described in "(2) Preparation of natural bagworm FibH solidified molded product" of Example 1.

[0117] (4) Wide-angle X-ray diffraction (WAXD) measurement This was performed in the same manner as in "(4) Wide-angle X-ray diffraction (WAXD) measurement" of Example 1.

[0118] (Results) The results are shown in Figure 2. The graph pattern is almost the same as that of the wide-angle X-ray diffraction results of the regenerated bagworm FibH cast film derived from natural bagworm FibH shown as A in Example 1 in Figure 1. This demonstrates that even artificial FibH, such as mutant bagworm FibH, can have α-helices and β-sheet structures in the molecule of the regenerated polypeptide molded product if it is α-βt / s coexisting FibH.

[0119] <Example 3: Confirmation of the structure of natural bagworm FibH in an aqueous solution> (Objective) To confirm that natural bagworm FibH, which is α-βt / s coexisting FibH, exists only in aqueous solution as an aqueous solution of α-βt coexisting FibH, which has a structure in which α-helix and β-turn coexist within the molecule.

[0120] (Method) Structural analysis of natural bagworm moth FibH in aqueous solution was performed by immobilizing natural bagworm moth FibH via freeze-drying. (1) Preparation of natural bagworm moth FibH aqueous solution The process was carried out in the same manner as in "(1) Preparation of natural bagworm moth FibH aqueous solution" of Example 1.

[0121] (2) Production of solidified molded article of natural bagworm moth FibH The natural bagworm moth FibH aqueous solution obtained in (1) was freeze-dried at -80°C using a freeze dryer (manufactured by EYELA) to obtain a solidified molded article of natural bagworm moth FibH.

[0122] (3) Wide-angle X-ray diffraction (WAXD) measurement The measurement was performed in the same manner as in "(4) Wide-angle X-ray diffraction (WAXD) measurement" of Example 1.

[0123] (Results) The results are shown in Figure 3. For natural bagworm moth FibH in aqueous solution, the α 10-10 plane reflection and Silk-I 101 plane reflection were confirmed. Silk-I 101 is a water-soluble crystal structure composed of aggregates of β-turns. Therefore, it was revealed that natural bagworm moth FibH exists as α-βt coexisting FibH in which α-helix and β-turn coexist within the molecule in aqueous solution.

[0124] <Example 4: Verification of structure and water solubility of regenerated FibH molded articles derived from various silk-spinning insects> (Objective) The structures of various solidified FibH molded articles produced from FibH aqueous solutions derived from bagworm moths and other silk-spinning insects (insects that spin silk) are comparatively verified by wide-angle X-ray diffraction, and the solubility of each solidified molded article in water is confirmed.

[0125] (Method) (1) Preparation of various natural FibH aqueous solutions Eumeta japonica, Bombyx mori, and Samia cynthia ricini were used as the silk-spinning insects.

[0126] a. Preparation of natural bagworm moth FibH aqueous solution The process was carried out in the same manner as in "(1) Preparation of natural bagworm moth FibH aqueous solution" of Example 1.

[0127] b. Preparation of Natural Silkworm FibH Aqueous Solution Silk threads obtained from silkworm cocoons by reeling were scouring with a sodium carbonate aqueous solution to obtain natural silkworm FibH. The obtained silkworm FibH was treated with a lithium bromide aqueous solution to dissolve it. Subsequently, it was placed in a cellulose dialysis tube and desalted to obtain a natural silkworm FibH aqueous solution.

[0128] c. Preparation of natural Eri silkworm FibH aqueous solution: After gut purging, the final instar larvae of Eri silkworms were anesthetized on ice, and the dorsal side was incised to extract the silk glands using forceps. Subsequently, the posterior silk glands were removed and immersed in pure water cooled to 4°C for approximately 24 hours to obtain an Eri silkworm FibH aqueous solution consisting only of fibroin H chains.

[0129] (2) Preparation of natural FibH cast films (FibH solidified molded products) of various FibH The various FibH aqueous solutions obtained in (1) above were cast onto plastic petri dishes and dried in a 20°C environment to obtain cast films as FibH solidified molded products.

[0130] (3) Wide-angle X-ray diffraction (WAXD) measurement The various recycled natural FibH cast films obtained in (2) above were subjected to the same method as in "(4) Wide-angle X-ray diffraction (WAXD) measurement" of Example 1.

[0131] (4) Dissolution tests in water for various recycled FibH cast films A total of four types of FibH cast films were tested for solubility in water: the three types of recycled natural FibH cast films obtained in (2), and the cast film of recycled mutant bagworm FibH (artificial bagworm FibH) obtained in Example 2 "(3) Preparation of mutant bagworm FibH solidified molded product".

[0132] Using a red oil-based marker, mark approximately 10 x 10 mm on each film. 2 A square was drawn, and the film was cut into a square shape using a utility knife to create the test specimen.

[0133] Various test specimens were immersed in plastic petri dishes filled with pure water, and their solubility in water over time was tracked. Specifically, at the start of the test, the test specimens were completely immersed in water for approximately 10 seconds using tweezers to wet both sides of the specimens, and then the specimens that floated to the water surface were observed in that state over time.

[0134] (Results) Figure 4 shows the wide-angle X-ray diffraction results, and Table 1 shows the water solubility test results.

[0135]

[0136] In Figure 4, silkworm A shows a diffraction peak indicating the presence of Silk-I type crystals, which are aggregates of β-turn structures (Silk-I 002 / 110 Surface reflection was observed. In addition, in Erisan B, a diffraction peak (α) indicating α-helix aggregate formation was observed. 10-10 Surface reflection was confirmed. And in the bagworm C, as shown in Example 1, diffraction peaks (α) showing the coexistence of α-helix aggregates and β-sheet crystals were observed. 10-10 Surface reflection, and β 200 / 210 Surface reflection and β 211 Surface reflection was confirmed.

[0137] Furthermore, the results in Table 1 demonstrate that only the regenerated FibH coagulated molded product derived from bagworm FibH, which is α-βt / s coexisting FibH, becomes insoluble regardless of whether it is natural FibH or mutant FibH.

[0138] <Example 5: Verification of physical properties of recycled FibH molded products with and without insolubilization treatment> (Objective) To compare the physical properties of the recycled FibH cast film without insolubilization treatment of the present invention with those of a conventional FibH cast film that has undergone insolubilization treatment.

[0139] (Method) (1) Preparation of natural FibH aqueous solution The silkworms used were the large bagworm and the silkworm. The preparation of natural FibH aqueous solution for each species was carried out in accordance with the method described in Example 4, "(1) Preparation of various natural FibH aqueous solutions".

[0140] (2) Preparation of cast films (molded products) of various FibH The method for preparing cast films from aqueous solutions of each type of natural FibH was in accordance with the method described in Example 4, "Preparation of regenerated natural FibH cast films (FibH solidified molded products) of various FibH".

[0141] (3) Immobilization treatment of silkworm-recycled FibH cast film As shown in the results of Example 4, silkworm-recycled FibH cast film is soluble in water and therefore requires immobilization treatment. Therefore, the silkworm-recycled FibH cast film obtained in (2) was immersed in 80% ethanol at 20°C for 6 hours to perform immobilization treatment. After that, it was air-dried for 24 hours. Subsequently, a portion of the prepared film was cut out as a test piece and completely immersed in water for more than 24 hours to confirm that it had become insoluble in water. Note that the bagworm-recycled FibH cast film was not subjected to immobilization treatment.

[0142] (4) Tensile Test of Recycled FibH Cast Film 20mm x 2mm test pieces were cut from silkworm recycled FibH cast film that had been immobilized and from bagworm recycled FibH cast film that had not been immobilized, and the film thickness, width, and gripping distance of the test pieces were measured. Film thickness was measured using a Digimicro MF-501 (Nikon), and tensile testing was performed using a desktop precision universal testing machine AUTOGRAPH AGS-X 5kN (Shimadzu Corporation) with an attached 50N load cell at a tensile speed of 1.0mm / min. Measurements and tests were performed at room temperature (approximately 25°C, 60% humidity).

[0143] Stress was calculated by dividing the force value obtained from the tensile test by the cross-sectional area of ​​the sample before tensioning (product of film thickness and width). Strain was calculated by dividing the displacement obtained from the tensile test by the working length (distance between grips) before tensioning.

[0144] (Results) The results are shown in Figure 5. In the figure, the solid line (a) represents the silkworm-regenerated FibH cast film that has been immobilized, and the dashed line (b) represents the bagworm-regenerated FibH cast film that has not been immobilized.

[0145] The immobilized silkworm-recycled FibH cast film (a) fractured at a maximum point stress of 70 MPa, and the maximum point displacement strain was only 3%. Here, "maximum point stress" refers to the stress just before fracture. Generally, a larger value means that it can withstand stronger stress, i.e., higher strength. Also, "maximum point displacement strain" refers to the elongation at fracture, which is the elongation of the sample until it fractures. Generally, a larger value means that it elongates well. In other words, this result indicates that the immobilized silkworm-recycled FibH cast film exhibits the same embrittlement of the recycled polypeptide due to the immobilization treatment as in conventional problems.

[0146] On the other hand, in the untreated bagworm-recycled FibH cast film, the maximum point stress was 67 MPa, but no fracture occurred, and the maximum point displacement strain reached 12%. This result indicates that the bagworm-recycled FibH cast film, despite not undergoing immobilization treatment, possesses comparable strength to the immobilized silkworm-recycled FibH cast film, and exhibits significantly improved elongation, i.e., ductility, without becoming brittle. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A fibroin H-chain aqueous solution for processing polypeptide molded articles, comprising a fibroin H-chain protein containing one or more α-helix-forming sequences and β-turn-forming sequences in one molecule, 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 in a polypeptide coagulated molded article obtained by dehydrating the aqueous solution, and 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 a polypeptide coagulated molded article obtained by dehydrating the aqueous solution.

2. The aqueous solution according to claim 1, 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 GGX (where X is A or S) or SEQ ID NO:

2.

3. The aqueous solution according to claim 1 or 2, wherein the β-turn forming sequence consists of GXGXGX (where X is A or S), or the amino acid sequence shown in SEQ ID NO: 3, and / or the amino acid sequence shown in SEQ ID NO:

4.

4. The aqueous solution according to any one of claims 1 to 3, wherein all or part of the fibroin H chain protein is a mutant fibroin H chain protein.

5. The aqueous solution according to any one of claims 1 to 4, wherein the fibroin H chain protein is derived from bagworm silk.

6. A polypeptide coagulated molded article obtained by dehydrating an aqueous solution according to any one of claims 1 to 5.

7. A polypeptide molded article obtained by molding a polypeptide solidified molded article according to claim 6.

8. A method for producing a polypeptide molded article that does not require insolubilization treatment, comprising a coagulation step of dehydrating and coagulating an aqueous solution of fibrous protein for processing polypeptide molded articles to obtain a polypeptide coagulated molded article, wherein the aqueous solution of fibrous protein is an aqueous solution containing a fibrous protein that each contains one or more α-helix-forming sequences and β-turn-forming sequences in one molecule, 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 in the polypeptide coagulated molded article obtained by dehydrating the aqueous solution, and the β-turn-forming sequence consists of an amino acid sequence that forms a β-sheet structure 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 obtained by dehydrating the aqueous solution.

9. The manufacturing method according to claim 8, comprising a molding step of shaping a polypeptide solidified molded product into a desired shape.

10. The manufacturing method according to claim 8 or 9, 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 GGX (where X is A or S) or SEQ ID NO:

2.

11. The manufacturing method according to any one of claims 8 to 10, wherein the β-turn forming sequence consists of GXGXGX (where X is A or S), or the amino acid sequence shown in SEQ ID NO: 3 and / or the amino acid sequence shown in SEQ ID NO:

4.

12. The manufacturing method according to any one of claims 8 to 11, wherein the fibrous protein is a fibroin H chain protein.

Images