Terminal region mutant silk protein, composition, and silk protein aqueous solution

Abstract

Provided are: a terminal region mutant silk protein obtained by introducing a mutation into the amino acid sequence of the N-terminal region and / or the C-terminal region of a silk protein; and a silk protein aqueous solution comprising the same. At least 70% of amino acids constituting a central region positioned between the N-terminal region and the C-terminal region may be composed of glycine residues and alanine residues.

Description

Terminal Region Mutant Silk Protein, Composition and Aqueous Silk Protein Solution

[0001] The present invention relates to a terminal region mutant silk protein, a composition comprising the terminal region mutant silk protein, an aqueous silk protein solution, and a method for suppressing gelation of an aqueous silk protein solution. The present application claims priority based on Japanese Patent Application No. 2025-030970 filed with Japan on February 28, 2025, and International Application No. PCT / JP2025 / 007052 filed internationally on February 28, 2025, the contents of which are incorporated herein by reference.

[0002] In recent years, toward the realization of a sustainable society, there has been a demand for the development of environmentally friendly and highly safe alternatives to synthetic resins typified by plastics. For example, polypeptide molded products using proteins and the like as raw materials are one such alternative. Among them, polypeptide molded products derived from silk proteins produced by organisms such as insects and spiders combine biodegradability and high biocompatibility in addition to the intrinsic strength, elongation, and high toughness of natural silk, and therefore are attracting attention as a material in fields such as medical materials and structural materials.

[0003] Polypeptide molded products are generally produced using liquid protein as a raw material, followed by coagulation and molding. For example, in the case of a polypeptide molded product made of silk protein, the raw material is an aqueous silk protein solution obtained by dissolving fiber-formed silk in a solvent to obtain a liquid state, or a liquid silk protein obtained by cloning a gene encoding silk protein and expressing the same in Escherichia coli or the like.

[0004] Accordingly, after silk protein is liquefied, it needs to be maintained in a liquid state for a certain period of time through the steps of dialysis, concentration, and storage until it is subjected to molding processing.

[0005] However, silk protein is unstable in liquid form, especially in aqueous solutions, and has a tendency to gel accidentally during various processes such as preparation, storage, and processing. Therefore, handling it in liquid form is difficult, and it cannot be maintained in a liquid state for extended periods. This has hindered the stable molding and processing of molded products using silk protein aqueous solutions as raw materials, and has been a contributing factor to the delay in their practical application.

[0006] To solve the above problems, various methods have been developed to reduce the frequency of gelation, such as reducing the gelation rate or delaying the gelation time. Examples include static storage with vibration isolation, low-temperature storage, and pH control (Non-Patent Literature 1 and Non-Patent Literature 2). Furthermore, efforts have been made to achieve synergistic effects by combining multiple of these methods. However, none of these methods can completely suppress gelation, nor can they withstand long-term storage exceeding one month.

[0007] Shinichiro Hiraide, 2007, Nagano Prefectural Industrial Technology Center Research Report 2:I36-I39. Matsumoto A., et al., 2006, J. Phys. Chem., 110:21630-21638.

[0008] The objective of this invention is to develop and provide a method that can suppress the gelation of liquid silk proteins for an extended period of time.

[0009] To solve the above problems, the inventors did not investigate methods or conditions for suppressing the gelation of silk protein aqueous solutions as in the conventional approach, but instead focused on the silk protein itself. Specifically, they attempted to develop mutant silk proteins that do not gel, or gel less easily, even when in a liquid state, by modifying the silk protein.

[0010] The inventors created mutant silk proteins by introducing various mutations into the amino acid sequence of silk protein and investigated the gelation-inhibiting effect of each aqueous solution. As a result, they found that aqueous solutions with mutations introduced in the N-terminal and C-terminal regions showed a significant reduction in the rate of gelation. Furthermore, it was revealed that when stored at low temperatures, gelation could be suppressed for more than one month.

[0011] In recent years, it has become increasingly clear that the N-terminal and C-terminal regions of spider silk proteins contribute to the fiber structure formation mechanism. For example, Malay et al. (AD et al., 2020, Sci. Adv. 6, ebb6030) have disclosed a self-assembling fibril formation mechanism based on the pH responsiveness of the N-terminal region and the metal ion responsiveness of the C-terminal region in silk proteins. Furthermore, it has been revealed that the amino acid sequences of the N-terminal and C-terminal regions of silk proteins are similar among species belonging to the order Lepidoptera. However, the mechanism of gelation of the N-terminal and C-terminal regions in aqueous solutions of silk proteins has not been clarified until now. One aspect of the present invention is based on novel findings from the above development results and includes the following.

[0012] (1) A terminal region mutant silk protein, wherein the protein is a fibroin protein or a spidoin protein comprising an N-terminal region, a central region, and a C-terminal region in that order from the N-terminus, and the entirety of either the N-terminal region and the C-terminal region, and / or one or more amino acid mutations of addition, deletion, or substitution in either the N-terminal region and the C-terminal region. (2) The terminal region mutant silk protein according to (1), wherein the N-terminal region is (a) an amino acid sequence shown in any of SEQ ID NOs: 1 to 18 and 95 to 96, or (b) an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 18 and 95 to 96, and 70% or more of the amino acids constituting the central region consist of glycine residues and alanine residues, and the C-terminal region consists of (c) an amino acid sequence shown in any of SEQ ID NOs: 37 to 54 and 99 to 103, or (d) an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in any of SEQ ID NOs: 37 to 54 and 99 to 103. (3) The terminal region mutant silk protein according to (1) or (2), wherein the fibroin protein is derived from a bagworm or a silkworm. (4) A composition comprising any one or more terminal region mutant silk proteins according to (1) to (3). (5) An aqueous solution of silk protein containing one or more terminal region mutant silk proteins as described in (1) to (3). (6) A method for suppressing gelation of an aqueous solution of silk protein, comprising a heating step of heating the aqueous solution of silk protein described in (5) at 0°C to 40°C.

[0013] According to the terminal region mutant silk protein of the present invention or a composition containing the same, an aqueous solution of silk protein that is less likely to gel or does not gel at all can be prepared by dissolving it in water.

[0014] The present invention provides a silk protein aqueous solution that is less prone to gelation and can be maintained in a liquid state for a long period of time.

[0015] According to the present invention's method for suppressing gelation of an aqueous solution of silk protein, gelation of the aqueous solution of silk protein can be suppressed for a long period of time.

[0016] This is a conceptual diagram showing the basic structure of wild-type silk protein. This is a conceptual diagram showing the bw753 protein, a mutant fibroin H chain protein (hereinafter also referred to as "FibH") derived from bagworms. This is a conceptual diagram showing the bw753ΔC protein, which has the entire C-terminal region of the bw753 protein shown in Figure 2A deleted. This is a conceptual diagram showing the bw753ΔN protein, which has the entire N-terminal region of the bw753 protein shown in Figure 2A deleted. This is a conceptual diagram showing the bw753ΔN / C protein, which has both the N-terminal and C-terminal regions of the bw753 protein shown in Figure 2A deleted. These are CBB staining and Western blotting diagrams of each mutant protein expressed in E. coli in Example 1. In the diagrams, M indicates a marker lane. In each diagram, the band indicated by the arrow is the target mutant protein.

[0017] Unless otherwise specified, "a," "an," and "the" are understood to mean "one or more," encompassing both singular and plural forms.

[0018] The term "comprise" means that it may include components other than the component being discussed. The term "consist of" means that it does not include components other than the component being discussed. The term "consistently of" means that it does not include components other than the component being discussed in a manner that performs a special function (such as a manner that completely negates the effect of the invention). In this specification, when "comprise" is used, it includes the "consist of" and "consistently of" manners.

[0019] A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0020] If multiple upper and lower limits are specified for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range.

[0021] 1. Terminal Region Mutant Silk Protein 1-1. Overview The first aspect of the present invention is a terminal region mutant silk protein. The protein of this aspect is either completely deleted from either the N-terminal region or the C-terminal region, or / or contains one or more amino acid mutations of addition, deletion, or substitution in either or both of them. The terminal region mutant silk protein of this aspect functions as a poor gelling agent or as an active ingredient in the poor gelling composition described later, and by dissolving it in water, an aqueous solution of silk protein that is less likely to gel can be easily prepared.

[0022] 1-2. Definitions of Terms The following terms used in this invention are defined below.

[0023] In this specification, "molded article" refers to a solid body having 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.

[0024] In this specification, "polypeptide molded article" means a molded article containing a polypeptide such as a protein as its main component. It may also be composed solely of polypeptides. Specific examples of polypeptide molded articles include fibers, films, and plates. In this specification, unless otherwise specified, "polypeptide molded article" means a recycled polypeptide molded article.

[0025] In this specification, "regenerated polypeptide" refers to a polypeptide obtained by coagulating an aqueous polypeptide solution, thereby regenerating the liquid polypeptide dissolved in the solution into a solid polypeptide. In this specification, this mainly refers to regenerated silk protein obtained by regenerating an aqueous solution of silk protein.

[0026] In this specification, "recycled polypeptide molded article" means a molded article containing a recycled polypeptide, or a molded article consisting of a recycled polypeptide.

[0027] In this specification, "silk" refers to a protein-based thread produced by insects or arachnids for purposes such as nesting, movement, anchoring, cocooning, and prey capture. In this specification, when simply referred to as "silk," the name of the organism from which it originates is generally not specified, and it refers to a broad category of silk. When referring to silk from a specific organism, the name of that organism shall be placed before "silk," for example, silkworm silk or bagworm silk.

[0028] In this specification, "silk protein" refers to the high molecular weight fibrous protein that constitutes silk thread. This includes both wild-type silk protein and mutant silk protein. The basic structure of silk protein is described in detail in chapter 1-3, "Structure of Silk Protein." Specific examples of silk protein include fibroin protein and spidoin protein.

[0029] In this specification, "wild-type silk protein" refers to naturally occurring silk protein encoded by the wild-type silk protein gene of various organisms.

[0030] In this specification, "mutant silk protein" refers to a silk protein in which all or part of a wild-type silk protein has been artificially modified using genetic engineering or other technologies. Mutant silk proteins consist of an amino acid sequence different from that of wild-type silk proteins and, in principle, do not exist in nature. However, it is assumed that mutant silk proteins include the N-terminal region, the central region, and the C-terminal region, in order from the N-terminus, which are the basic components of wild-type silk proteins. Examples of mutant silk proteins include silk proteins in which one or more amino acids have been added, deleted, and / or substituted into the amino acid sequence of a silk protein, and chimeric silk proteins (hybrid silk proteins) in which the amino acid sequences of two or more different insect-derived silk proteins have been fused. Mutant silk proteins may have the same physical properties as wild-type silk proteins or they may have different physical properties.

[0031] In this specification, "fibroin protein" refers to the silk protein that constitutes silk threads derived from insects such as silkworms and bagworms. When simply referred to as fibroin protein, it includes both wild-type and mutant varieties. When referred to as fibroin protein in this specification, unless otherwise specified, it refers to fibroin H-chain protein.

[0032] In this specification, "fibroin H chain protein" refers to the main fibrous protein that constitutes the fibroin complex (silk fibroin elementary unit; SFEU complex). Generally, FibH has an amino acid sequence consisting of repeating glycine and alanine residues.

[0033] In this specification, "spidoin protein" refers to the silk protein that constitutes silk threads, particularly dragline silks, derived from organisms of the order Araneae or Acari. When simply referred to as "spidoin protein," it includes both wild-type and mutant forms. Its basic structure is similar to that of fibroin protein, and it has an amino acid sequence that includes many glycine and alanine residues.

[0034] In this specification, "gelation" refers to the process in which a substance dissolved in an aqueous solution (in this specification, silk protein) loses its fluidity, causing the viscosity of the aqueous solution to increase and resulting in the formation of a jelly-like solid.

[0035] In this specification, "difficult to gel" means that gelation does not occur easily, or that gelation hardly occurs at all.

[0036] In this specification, "silkworm" refers to the larva of the silkworm moth (Bombyx mori), belonging to the family Bombycidae in the order Lepidoptera. This insect has been used industrially under human management since ancient times for the production of silk thread from its cocoon. In recent years, it has also been used as a host for mass production systems of useful proteins other than silk through genetic engineering technology.

[0037] In this specification, "bagworm" refers to the larvae of moths belonging to the family Psychidae in the order Lepidoptera. It can refer to the larvae of various Psychidae moths, but the types of bagworms referred to here 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 here may belong to any of these genera. Specific examples of bagworm species include the large bagworm (Eumeta japonica) and the small bagworm (Eumeta minuscula).

[0038] In this specification, "Araneae organisms" refers to organisms belonging to the order Araneae. While this includes numerous species, the types of spiders described herein are not limited, however, web-building spiders that spin webs are preferred over wandering spiders. For example, species belonging to the superfamily Araneoidea are included. Specifically, for example, species belonging to the family Araneidae (such as Araneus ventricosus, Araneus diadematus, Argiope amoena, and Caerostris darwini), and species belonging to the family Nephilidae (such as Nephila pilipes, Nephila clavata, and Nephila clavipes).

[0039] As used herein, the term "silk protein gene" refers to a gene encoding a silk protein. Examples include the fibroin H chain (FibH) gene and the spidroin gene.

[0040] As used herein, the term "terminal region-modified silk protein" refers to a modified silk protein having a mutation in the N-terminal region and / or C-terminal region of a silk protein. The basic structure of the terminal region-modified silk protein is described in detail in the section "1-3. Structure of Silk Protein".

[0041] 1-3. Structure of Silk Protein The terminal region-modified silk protein of the present embodiment is characterized by comprising a mutation in the terminal region of a wild-type silk protein. Hereinafter, the structures of the wild-type silk protein and the terminal region-modified silk protein of the present embodiment will be described.

[0042] 1-3-1. Structure of Wild-Type Silk Protein As used herein, the term "wild-type silk protein" refers to a silk protein whose full-length amino acid sequence exists in nature. As described above, silk proteins herein include fibroin proteins and spidroin proteins. Therefore, a wild-type silk protein refers to a wild-type fibroin protein or a wild-type spidroin protein.

[0043] As shown in FIG. 1, a wild-type silk protein (0100) comprises, as a basic structure, an N-terminal region (0101), a central region (0102), and a C-terminal region (0103) in this order from the N-terminal side. This basic structure is common to all silk proteins regardless of the species of organism. In addition, many silk proteins further comprise a signal peptide (0104) on the N-terminal side of the N-terminal region. Hereinafter, each region will be specifically described.

[0044] (1) N-terminal region The "N-terminal region" (0101), in the amino acid sequence constituting a wild-type silk protein, refers to a region consisting of 70 to 250 amino acid residues that is located on the N-terminal side of the below-described central region as shown in FIG. 1 and does not contain any repeat units.

[0045] Specific examples of N-terminal amino acid sequences include those shown in SEQ ID NOs: 1 to 18. SEQ ID NO: 1 is the N-terminal amino acid sequence of FibH derived from the bagworm (Eumeta japonica). SEQ ID NO: 19 is an example of the nucleotide sequence encoding it. SEQ ID NO: 2 is the N-terminal amino acid sequence of FibH derived from the silkworm (Bombyx mori). SEQ ID NO: 20 is an example of the nucleotide sequence encoding it. SEQ ID NO: 3 is the N-terminal amino acid sequence of FibH derived from the Japanese silkworm (Antheraea yamamai), and SEQ ID NO: 21 is an example of the nucleotide sequence encoding it. SEQ ID NO: 4 is the N-terminal amino acid sequence of FibH derived from the silkworm (Antheraea pernyi), and SEQ ID NO: 22 is an example of the nucleotide sequence encoding it. SEQ ID NO: 5 is the N-terminal amino acid sequence of FibH derived from the wax moth (Galleria mellonella), and SEQ ID NO: 23 is an example of the nucleotide sequence encoding it. Sequence IDs 6-15 are the amino acid sequences of the N-terminal region of the spidoin protein derived from the orb-weaver spider (Araneus ventricosus), and examples of the encoding sequences include Sequence IDs 24-33, respectively. Sequence IDs 16-18 are the amino acid sequences of the N-terminal region of the spidoin protein derived from the golden orb-weaver spider (Nephila pilipes), and examples of the encoding sequences include Sequence IDs 34-36, respectively. Sequence IDs 95 and 96 are the amino acid sequences of the N-terminal region of the spidoin protein derived from the American golden orb-weaver spider (Nephila clavipes), and examples of the encoding sequences include Sequence IDs 97 and 98, respectively.

[0046] The N-terminal region of the present embodiment is not limited to the above specific examples. For example, an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the amino acid sequences represented by SEQ ID NOs: 1 to 18 and 95 to 96 falls under this category.

[0047] As used herein, the term "amino acid identity" refers to the percentage (%) of identical amino acids in one polypeptide relative to the total number of amino acids of the other polypeptide when the amino acid sequences of two polypeptides are aligned, and gaps are introduced into either amino acid sequence as necessary to maximize the degree of amino acid matching between the two. This percentage of amino acid identity can be easily determined by using a known program such as the homology search program BLAST (Basic local alignment search tool; Altschul, S.F. et al, J. Mol. Biol., 215, 403-410, 1990).

[0048] (2) Central Region The "central region" (0102) is a region responsible for the physical properties of silk protein as a fiber in each biological species, and is constituted by linking a plurality of repeating units consisting of identical and / or different amino acid sequences. The central region can be described as a region located between an N-terminal region that does not contain repeating units and a C-terminal region that does not contain repeating units, and is positioned on the C-terminal side of the N-terminal region and on the N-terminal side of the C-terminal region. The repeating units in the central region are directly linked to each other, but they may also be indirectly linked via a linking region interposed between the repeating units.

[0049] Although the amino acid sequence of the central region varies depending on the silk protein from each biological species, 70% or more, 72% or more, 74% or more, 76% or more, 78% or more, 80% or more, 82% or more, 84% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more of the sequence is composed of glycine residues and alanine residues.

[0050] A "Central Repeat Unit" (CRU) (0105) is the main constituent unit of the central region. A single repeat unit comprises multiple glycine residues and / or alanine residues. In addition, although there are some differences among species, many species contain glycine / alanine units and / or alanine clusters, and may also contain non-glycine / alanine moieties.

[0051] A "glycine / alanine unit (G / A unit)" is a subunit of a repeat unit composed of two amino acid residues: a glycine (Gly: G) residue and an alanine (Ala: A) residue, consisting of either glycine-alanine (GA) or alanine-glycine (AG). In silk proteins of many species, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the repeat unit is composed of G / A units.

[0052] An "alanine cluster" is a subunit of a repeating unit consisting of consecutive alanine (Ala) residues, and is found as a single cluster at the N-terminus of the silk protein of some species, such as bagworms. The Ala cluster contributes to the characteristic physical properties of the silk protein of that species. A single Ala cluster contains 15 to 25 alanine residues, but it may also contain other residues besides alanine, such as glutamic acid (Glu:E) residues or glutamine (Gln:Q) residues around the 10th position from the N-terminus of the Ala cluster.

[0053] A "non-glycine / alanine site (non-G / A site)" is a repeating unit consisting of 5 to 7 amino acid sequences other than glycine and alanine residues, and includes sites other than G / A units and Ala clusters. The amino acid residues that make up a non-G / A site are not limited and include, for example, serine (Ser:S) residues, valine (Val:V) residues, and tyrosine (Tyr:Y) residues.

[0054] As specific examples of repeating unit amino acid sequences, though not limited to them, sequence numbers 73-83 can be cited. Here, sequence numbers 73-81 are the amino acid sequences of repeating units in FibH derived from the bagworm (Philadelphia japonica). Also, sequence numbers 82 and 83 are the amino acid sequences of repeating units in FibH derived from the silkworm.

[0055] The "linking region" (0106) is an amino acid region that intervenes between each repeating unit constituting the central region and links them together, and consists of 5 to 50 amino acids, 8 to 40 amino acids, or 10 to 30 amino acids.

[0056] (3) C-terminal region The "C-terminal region" (0103) is a region consisting of 30 to 60 amino acid residues located on the C-terminal side of the central region (0102) in the amino acid sequence that constitutes the wild-type silk protein, as shown in Figure 1, and does not contain a repeating unit.

[0057] Specific examples of C-terminal amino acid sequences include those shown in SEQ ID NOs: 37-54 or 99-103. SEQ ID NO: 37 is the C-terminal amino acid sequence of FibH derived from the bagworm (Pagurus japonica). SEQ ID NO: 55 is an example of the nucleotide sequence encoding it. SEQ ID NO: 38 is the C-terminal amino acid sequence of FibH derived from the silkworm (Pagurus japonica). SEQ ID NO: 56 is an example of the nucleotide sequence encoding it. SEQ ID NO: 39 is the C-terminal amino acid sequence of FibH derived from the Japanese oak silkworm (Antheraea yamamai), and SEQ ID NO: 57 is an example of the nucleotide sequence encoding it. SEQ ID NO: 40 is the C-terminal amino acid sequence of the FibH protein derived from the succedanea moth, and SEQ ID NO: 58 is an example of the nucleotide sequence encoding it. SEQ ID NO: 41 is the C-terminal amino acid sequence of FibH derived from the wax moth (Gluteus scoparius), and SEQ ID NO: 59 is an example of the nucleotide sequence encoding it. Sequence IDs 42-51 are the amino acid sequences of the C-terminal region of the spidoin protein derived from the orb-weaver spider, and examples of the nucleotide sequences encoding them include Sequence IDs 60-69, respectively. Sequence IDs 52-54 are the amino acid sequences of the C-terminal region of the spidoin protein derived from the golden orb-weaver spider, and examples of the nucleotide sequences encoding them include Sequence IDs 70-72, respectively. Sequence IDs 99-101 are the amino acid sequences of the C-terminal region of the spidoin protein derived from the garden orb-weaver spider (Araneus diadematus), and examples of the nucleotide sequences encoding them include Sequence IDs 104-106, respectively. Sequence IDs 102 and 103 are the amino acid sequences of the C-terminal region of the spidoin protein derived from the American golden orb-weaver spider, and examples of the nucleotide sequences encoding them include Sequence IDs 107 and 108, respectively.

[0058] The C-terminal region of this embodiment is not limited to the above specific examples. For example, it includes amino acid sequences having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% 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: 37-54 and 99-103.

[0059] (4) Signal Peptides A "signal peptide" (signal sequence) (0104) is an extracellular translocation signal necessary for the secretion of proteins biosynthesized by gene expression into the extracellular space. After translation, signal peptides are cleaved and removed by signal peptidases before being secreted into the extracellular space. Signal peptides have a positively charged amino acid such as lysine or arginine at the N-terminus, followed by a sequence of highly hydrophobic amino acids such as alanine, leucine, valine, isoleucine, and phenylalanine.

[0060] Specific examples of amino acid sequences of signal peptides in silk proteins include the amino acid sequences shown in SEQ ID NOs. 84 is the amino acid sequence of the signal peptide in FibH derived from the bagworm (Pagurus japonica). SEQ ID NOs. 85 is the amino acid sequence of the signal peptide in FibH derived from the silkworm (Pagurus kobomugi). And SEQ ID NOs. 86 is the amino acid sequence of the signal peptide in the spidoin protein derived from the orb-weaver spider (Pagurus japonica).

[0061] 1-3-2. Composition of Terminal Region Mutant Silk Protein In this specification, "terminal region mutant silk protein" refers to a silk protein having mutations in the N-terminal region and / or C-terminal region of wild-type silk protein, as described above. Terminal region mutant silk protein and compositions containing it have the effect of being less prone to gelation or not gelling at all. Terminal region mutant silk protein refers to a polypeptide that functions as a poor-gelling agent in this embodiment or as an active ingredient in a poor-gelling composition in the second embodiment.

[0062] Terminal region mutant silk proteins are, in principle, mutant silk proteins that are based on the wild-type silk protein but with a portion of it artificially modified, and they consist of a different amino acid sequence from the wild-type silk protein.

[0063] The terminal region mutant silk protein of this embodiment may be obtained by introducing a mutation into a wild-type silk protein derived from a single species, or by introducing a mutation into a chimeric silk protein derived from multiple species. The latter example is a chimeric terminal region mutant silk protein in which the N-terminal region is derived from silkworm silk protein, the central region is derived from bagworm silk protein, and the C-terminal region is missing.

[0064] A. The basic structure of the terminal region mutant silk protein is the same as that of the wild-type silk protein. That is, it may include the N-terminal region, the central region, and the C-terminal region in that order from the N-terminus. However, the terminal region mutant silk protein is characterized by containing a mutation in either the N-terminal region or the C-terminal region, or both. Therefore, either the N-terminal region or the C-terminal region, or both, may be deleted, and these two regions are not essential components of the terminal region mutant silk protein. Furthermore, the terminal region mutant silk protein may include a signal peptide on the N-terminal side of the N-terminal region as needed, as well as other components such as a labeled peptide.

[0065] Figure 2A is a conceptual diagram showing the bw753 protein, a mutant FibH derived from bagworms. Figure 2B is a conceptual diagram showing the bw753ΔC protein, which has the entire C-terminal region of the bw753 protein shown in Figure 2A deleted. Figure 2C is a conceptual diagram showing the bw753ΔN protein, which has the entire N-terminal region of the bw753 protein shown in Figure 2A deleted. Figure 2D is a conceptual diagram showing the bw753ΔN / C protein, which has both the N-terminal and C-terminal regions of the bw753 protein shown in Figure 2A deleted. Below, we will explain each component of terminal-region mutant silk proteins using Figures 2A to 2D.

[0066] (1) N-terminal region (0201) The N-terminal region of the terminal region mutant silk protein is located on the N-terminal side of the central region, similar to the wild-type silk protein. In principle, this N-terminal region contains one or more amino acid mutations in the amino acid sequence of the N-terminal region of the wild-type silk protein mentioned above.

[0067] If the aforementioned amino acid mutation is a deletion, the entire N-terminal region may be completely deleted, or only a portion of it may be deleted, as shown in Figure 2C.

[0068] In this specification, "part" refers to one or two or more consecutive or discontinuous amino acids from the amino acid sequence constituting the terminal region (N-terminal region and / or C-terminal region), and which is less than the total number of amino acids. Preferably, this corresponds to the number of amino acids that can cause loss of function in the terminal region. For example, five or more, eight or more, ten or more, twelve or more, fifteen or more, eighteen or more, or twenty or more consecutive or discontinuous amino acids.

[0069] Furthermore, the amino acid mutation may involve 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.

[0070] In this specification, "multiple items" means two or more items, for example, 2 to 20 items, 2 to 15 items, 2 to 10 items, 2 to 7 items, 2 to 5 items, 2 to 4 items, or 2 to 3 items.

[0071] Furthermore, in this specification, amino acid substitutions are preferably conservative amino acid substitutions. This is because conservative amino acid substitutions can result in a structure or properties substantially equivalent to those of the wild-type protein.

[0072] Furthermore, the N-terminal region of a terminal-region mutant silk protein can have the same amino acid sequence as the N-terminal region of a wild-type silk protein that does not contain the mutation, but only if the C-terminal region, as described later, also contains a mutation.

[0073] (2) Central Region (0202) In terminal region mutant silk proteins, the central region is an essential component. Its basic structure is the same as that of the central region of the wild-type silk protein, so a detailed explanation is omitted.

[0074] On the other hand, since terminal-region mutant silk proteins are recombinant silk proteins, the central region may have any modified configuration, as long as it retains the basic structure of the central region that exhibits the physical properties of silk proteins. For example, mutations involving the addition, deletion, and / or substitution of one or more amino acids can be included, as long as the function of the central region is not lost.

[0075] Furthermore, the number of repeat units in the central region does not need to be the same as that of wild-type silk protein and can be arbitrarily selected. Usually, three or more are sufficient, and there is no upper limit. For example, it may contain five or more, eight or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, ninety or more, one hundred or more, one fifty or more, two hundred or more, two hundred or more, three hundred or more, three fifty or more, four hundred or more, four hundred or more, five hundred or more, eighty or more, or even one thousand or more. In the terminal region mutant silk proteins shown in Figures 2A to 2D, the central region containing four repeat units (0204) is illustrated.

[0076] (3) C-terminal region (0203) The C-terminal region of the terminal region mutant silk protein is located on the C-terminal side of the central region, similar to the wild-type silk protein. This C-terminal region contains, in principle, one or more amino acid mutations, such as additions, deletions, or substitutions, in the amino acid sequence of the C-terminal region of the wild-type silk protein described above, similar to the N-terminal region. For example, in the case of a deletion mutation, the entire C-terminal region may be completely deleted, as shown in Figure 2B, or only a part of it may be deleted. Furthermore, the terminal region mutant silk protein may have a configuration in which both the N-terminal and C-terminal regions are completely deleted, as shown in Figure 2D.

[0077] Furthermore, the C-terminal region of a terminal-region mutant silk protein can have the same amino acid sequence as the C-terminal region of a wild-type silk protein that does not contain the mutation, but only if the N-terminal region mentioned above is also mutant.

[0078] (4) Signal Peptide Although not shown in Figures 2A to 2D, the terminal region mutant silk protein of this embodiment may contain a signal peptide. The signal peptide is selective in composition and, as needed, is ligated to the N-terminal side of the N-terminal region, or to the N-terminal side of the central region if the N-terminal region is completely deleted. The specific composition of the signal peptide may be the same as that of the signal peptide of wild-type silk protein.

[0079] In terminal-region mutant silk proteins, the signal peptide may be an exogenous signal peptide in addition to the endogenous signal peptide described above. For example, when introducing and expressing a gene encoding a terminal-region mutant silk protein into a host cell such as E. coli, an exogenous signal peptide derived from the host (E. coli in this example) is preferred. The amino acid sequence of the exogenous signal peptide, or the nucleotide sequence encoding it, may be one of known sequences and is not limited to any particular sequence.

[0080] (5) Labeled Peptide (0205) The terminal region mutant silk protein of this embodiment may optionally contain a labeled peptide. A "labeled peptide" is a peptide that is expressed together with the target polypeptide (in this case, the terminal region mutant silk protein) and serves as an indicator for its detection or extraction without inhibiting or suppressing the activity of the target polypeptide. Typically, the labeled peptide is configured to be expressed together with the terminal region mutant silk protein in the form of a fusion polypeptide. The position of the labeled peptide is not limited, but it is usually positioned on the N-terminal and / or C-terminal side of the target polypeptide.

[0081] Examples of labeled peptides, though not limited to them, include histidine (His) tags (e.g., (His)6 to (His) 10 Examples include peptide tags such as FLAG tags, Myc tags, or HA tags, as well as GFP proteins.

[0082] B. Specific Examples of Terminal Region Mutant Silk Proteins Specific examples of terminal region mutant silk proteins in this embodiment include polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 89 to 91. SEQ ID NO: 89 is the amino acid sequence of the bw753ΔC protein, consisting of 721 amino acids, in which the C-terminal region is completely deleted, as shown in Figure 2B, from the bw753 protein (SEQ ID NO: 87), which is the mutant bagworm FibH shown in Figure 2A constructed in Example 1 described later. SEQ ID NO: 90 is the amino acid sequence of the bw753ΔN protein, consisting of 643 amino acids, in which the N-terminal region is completely deleted, as shown in Figure 2C, from the bw753 protein. Furthermore, SEQ ID NO: 91 is the amino acid sequence of the bw753ΔN / C protein, consisting of 611 amino acids, in which both the N-terminal and C-terminal regions are completely deleted, as shown in Figure 2D, from the bw753 protein.

[0083] As an example of a specific nucleotide sequence of the gene encoding the mutant bagworm FibH, the nucleotide sequence shown in SEQ ID NO: 88, which encodes the bw753 protein constructed in Example 1, is included. Furthermore, as examples of specific nucleotide sequences of the gene encoding the terminal region mutant silk protein in this embodiment, the nucleotide sequence shown in SEQ ID NO: 92, which encodes the bw753ΔC protein constructed in Example 1, the nucleotide sequence shown in SEQ ID NO: 93, which encodes the bw753ΔN protein, and the nucleotide sequence shown in SEQ ID NO: 94, which encodes the bw753ΔN / C protein, are included.

[0084] 2. Non-Gellable Composition 2-1. Overview The second aspect of the present invention is a non-gelable composition. In this specification, "non-gelable composition" means a composition that, when dissolved in an aqueous solution, can prepare a non-gelable silk protein aqueous solution. The non-gelable composition of this aspect is a composition in which the terminal region mutant silk protein described in the first aspect is an active ingredient, and by dissolving it in water or an aqueous solution, the silk protein aqueous solution described in the third aspect can be prepared. Furthermore, it has advantages in terms of preservation, transportability, and storage compared to the silk protein aqueous solution.

[0085] 2-2. Composition 2-2-1. Components The poorly gelling composition of this embodiment includes an active ingredient as an essential component and a carrier and / or solvent as optional components. Each component will be described in detail below.

[0086] (1) Active ingredient The poorly gelling composition of this embodiment contains the terminal region mutant silk protein described in the first embodiment as an active ingredient. The composition of the terminal region mutant silk protein is described in detail in the first embodiment, so a specific explanation is omitted here.

[0087] The terminal region mutant silk protein described in the first embodiment, included in the poorly gelling composition of this embodiment, may be one type or multiple types. For example, it may include a terminal region mutant silk protein derived from spiders and a terminal region mutant silk protein derived from bagworms.

[0088] The amount of the active ingredient contained in the non-gelling composition of this embodiment is not particularly limited, as long as it is an amount that can suppress the gelation of the aqueous solution of silk protein of the third embodiment when the non-gelling composition of this embodiment is dissolved in water or the like to prepare the aqueous solution, and that can be regenerated into regenerated silk protein by coagulation treatment. The amount of the active ingredient generally varies depending on the type of active ingredient, the dosage form, and the type of solvent and carrier, which are other components described later.

[0089] (2) Carrier The poor-gelling composition of this embodiment may contain any carrier, as long as it does not inhibit the gelling inhibitory effect of the aqueous solution of the silk protein of the third embodiment when the poor-gelling composition of this embodiment is dissolved in water or the like to prepare the aqueous solution of the silk protein of the third embodiment, and does not inhibit or suppress the regeneration of the regenerated silk protein by coagulation treatment.

[0090] Depending on the form and application of the poorly gelling composition, the carrier may contain excipients, additives, auxiliaries, and / or other components.

[0091] Excipients include, for example, pH adjusters, fluid additive regulators, and emulsifiers. Examples of pH adjusters include sodium hydroxide and potassium hydroxide as alkaline agents, and citric acid, glycolic acid, and ascorbic acid as acidic agents. Examples of fluid additive regulators include silicates, talc, stearate, or polyethylene glycol. Examples of emulsifiers include surfactants.

[0092] Examples of additives include UV absorbers, antioxidants, stabilizers, preservatives, desiccants, isotonic solutions, and disinfectants, natural or chemical pigments.

[0093] Other components include, for example, wild-type silk protein.

[0094] (3) Solvent If the poorly gelling composition of this embodiment is in a semi-solid state (gel state) or a solution state, it may contain a solvent. Water or an aqueous solution can be used as the solvent. In addition, when the poorly gelling composition of this embodiment is dissolved in water or the like to prepare the silk protein aqueous solution of the third embodiment, an organic solvent such as ethanol may be used, provided that it does not inhibit the gelling inhibitory effect of the aqueous solution and does not inhibit or suppress the regeneration of the regenerated silk protein by coagulation treatment, in a range and content that does not inhibit the regeneration of the regenerated silk protein by coagulation treatment.

[0095] 2-2-2. Form The form of the poorly gelling composition of this embodiment may be solid or liquid. In the case of a solid, it may be powder, granules, small lumps, or semi-solid. The form may be determined as appropriate considering factors such as shelf life, transportability, and storage.

[0096] 3. Silk Protein Aqueous Solution 3-1. Overview The third aspect of the present invention is a silk protein aqueous solution. The silk protein aqueous solution of this aspect is an aqueous solution obtained by dissolving the terminal region mutant silk protein described in the first aspect, or the non-gelling composition described in the second aspect, in a solvent such as water or an aqueous solution, and contains the terminal region mutant silk protein described in the first aspect. The silk protein aqueous solution of this aspect can be used as a raw material for regenerated silk protein and molded articles thereof, and compared to conventional silk protein aqueous solutions, it is possible to provide a non-gelling silk protein aqueous solution that can be maintained in a liquid state for a long period of time without causing gelation.

[0097] 3-2. Composition The aqueous silk protein solution of this embodiment contains, as essential components, the terminal region mutant silk protein described in the first embodiment and water. It may also contain the carrier and solvent included in the poor gelling composition described in the second embodiment.

[0098] The aqueous solution of silk protein may contain one or more types of terminal region mutant silk proteins.

[0099] The concentration of terminal-region mutant silk protein in the aqueous silk protein solution is not particularly limited, as long as it can suppress the progression of gelation compared to conventional aqueous silk protein solutions and can be regenerated as regenerated silk protein by dehydration treatment. Typically, the mass percentage of regenerated silk protein relative to the mass of the aqueous solution (hereinafter the same applies in this specification) should be 0.1% to 30%, 0.2% to 28%, 0.3% to 25%, 0.4% to 22%, 0.5% to 20%, 0.8% to 15%, 0.8% to 14%, 1% to 12%, 2% to 10%, 4% to 8%, or 3% to 6%.

[0100] 3-3. Preparation of aqueous solution The aqueous solution of silk protein according to this embodiment can be prepared by dissolving the terminal region mutant silk protein described in the first embodiment in water or an aqueous solution.

[0101] The method for preparing the aqueous solution of silk protein can be any method known in the field for preparing an aqueous solution of silk protein.

[0102] 3-4. Effects: Regenerated silk protein molded products are considered extremely useful as medical materials and other applications due to their environmental friendliness, biodegradability, high safety, and biocompatibility. However, the raw material, the aqueous silk protein solution, has a problem of accidentally gelling during various processes in which it is used. This problem is considered to be the reason why the practical application of regenerated silk protein molded products has been hindered.

[0103] However, the silk protein aqueous solution of this embodiment can remain in a liquid state for a long period of time, even at concentrations more than twice as high as conventional silk protein aqueous solutions. This resolves the conventional problems associated with silk protein aqueous solutions and makes it possible to advance the practical application of recycled silk protein molded products.

[0104] 4. Method for suppressing gelation of a silk protein aqueous solution 4-1. Overview The fourth aspect of the present invention is a method for suppressing gelation of a silk protein aqueous solution. According to the gelation suppression method of the present invention, by keeping the silk protein aqueous solution described in the third aspect warm under specific temperature conditions, gelation of the aqueous solution can be suppressed and it can be maintained in a liquid state for a long period of time.

[0105] 4-2. Process The method for suppressing gelation of the silk protein aqueous solution according to this embodiment includes a heat retention step as an essential step, and the heat retention step will be described below.

[0106] (1) Heat retention process The "heat retention process" is a process of keeping the silk protein aqueous solution warm within a predetermined temperature range. The temperature range for heat retention in this process may be 0 to 40°C, 0 to 35°C, 0 to 30°C, 0 to 25°C, 0 to 20°C, 1 to 18°C, 1 to 16°C, 2 to 14°C, 2 to 12°C, 3 to 10°C, 3 to 8°C, or 4 to 6°C.

[0107] The storage location is not particularly limited as long as it is an environment that can maintain a constant temperature as much as possible within the above temperature range. Usually, a refrigerator (around 4°C) or an incubator set to the above temperature range is sufficient. The storage condition at the above temperature is fine as long as it is kept still, but stirring is also acceptable if necessary.

[0108] The concentration of silk protein in the aqueous solution of silk protein used in this process may be within the concentration range described in "3-2. Composition" above.

[0109] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. <Example 1: Preparation of terminal region mutant silk protein> (Objective) To prepare terminal region mutant silk protein.

[0110] (Methods) The bagworm FibH gene was modified using genetic engineering techniques to prepare various mutant bagworm FibH genes encoding terminal region mutant silk proteins. Expression vectors containing these genes were introduced into E. coli and then expressed to obtain the desired terminal region mutant bagworm FibH.

[0111] (1) Preparation of the mutant bagworm FibH gene The mutant bagworm FibH gene was prepared 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. 88 and encodes the bw753 protein, which is a mutant bagworm FibH derived from the giant bagworm moth. The "bw753 (protein)" (Figure 2A) consists of 753 amino acids shown in Sequence ID No. 87 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] (2) Preparation of terminal region mutant bagworm FibH gene The "bw753ΔC gene" (SEQ ID NO: 92), which is a mutant bagworm FibH gene bw753 gene, has the base sequence encoding the C-terminal region deleted, the "bw753ΔN gene" (SEQ ID NO: 93), which has the base sequence encoding the N-terminal region deleted, and the "bw753ΔN / C gene" (SEQ ID NO: 94), which has the base sequences encoding both the N-terminal and C-terminal regions deleted, are used in Example 3 of WO2020 / 235692 The vectors were prepared according to the method disclosed. Expression vectors for each terminal region mutant bagworm FibH gene inserted into the expression vector pET-26b(+) (Novagen) were as follows: the bw753ΔC gene expression vector was pET-26b-bw753ΔC, the bw753ΔN gene expression vector was pET-26b-bw753ΔN, and the bw753ΔN / C gene expression vector was pET-26b-bw753ΔN / C, similar to Example 3 of WO2020 / 235692.

[0114] (3) Preparation of Escherichia coli transformants and expression of terminal region mutant bagworm FibH After purifying the terminal region mutant bagworm FibH gene expression vectors (pET-26b-bw753ΔC, pET-26b-bw753ΔN, and pET-26b-bw753ΔN / C) constructed in (2) above, Escherichia coli transformants were prepared by introducing them into cells of Escherichia coli BLR (DE3) strain (Novagen) using a standard method. For control, Escherichia coli transformants were prepared by similarly introducing pET-26b-bw753. The induction of protein expression in E. coli transformants and the extraction and purification of solubilized proteins were carried out according to the method disclosed in Example 4 of WO2024 / 057361, yielding the target terminal region mutant bagworm FibH "bw753ΔC (protein)" (Figure 2B), "bw753ΔN (protein)" (Figure 2C), and "bw753ΔN / C (protein)" (Figure 2D), as well as the control "bw753 (protein)" (Figure 2A).

[0115] Furthermore, to confirm the expression of each purified protein, each protein was subjected to electrophoresis on a 10% SDS-polyacrylamide gel, followed by CBB staining and Western blotting, according to the method disclosed in Example 4 of WO2024 / 057361. Since all proteins expressed from the aforementioned expression vectors have a His6 tag at the C-terminus, they were reacted with an HRP-labeled anti-His antibody (Anti-His-tag mAb-HRP-DirecT; MBL) according to the attached protocol. Subsequently, a luminescence reaction based on HRP activity was performed using the luminescent substrate Western Lightning Plus-ECL (Perkin Elmer), and detected using a luminescent image analyzer Amersham Imager 600 (GE Healthcare).

[0116] (Results) Figure 3 confirms that the expression and preparation of each terminal region mutant protein were carried out normally. In this example, terminal region mutant bagworm FibH was prepared in a solubilized liquid state.

[0117] <Example 2: Verification of gelation behavior of terminal region mutant silk protein in aqueous solution> (Objective) To verify the gelation behavior of terminal region mutant silk protein in aqueous solution.

[0118] (Method) For each terminal region mutant bagworm FibH prepared in Example 1, and for the control mutant bagworm FibH, 10 mL of each bagworm FibH aqueous solution with a concentration of 1% was prepared. These were placed in 50 mL sealed containers and stored in a refrigerator at 4°C without a lid.

[0119] As the volume of the aqueous solution decreased due to natural evaporation and the concentration of each bagworm FibH aqueous solution increased, the solution was left undisturbed in the refrigerator until the volume of the solution was reduced to 1 / 6 (until the concentration of each terminal region mutant bagworm FibH reached 6%). During this time, the presence or absence of gelation in each aqueous solution was checked by whether or not cracks formed when the tip of tweezers was inserted into the liquid surface. After reaching 6%, the container was sealed with a lid and the aqueous solution was stored again in a refrigerator at 4°C, and the presence or absence of gelation was evaluated once a day until gelation occurred.

[0120] (Results) The results are shown in Table 1.

[0121]

[0122] bw753, an aqueous solution of the mutant bagworm FibH, induced gelation at concentrations of 2-3%. On the other hand, in aqueous solutions of terminal-region mutant silk proteins, none of the bw753ΔC protein, bw753ΔN protein, or bw753ΔN / C protein induced gelation up to 6%, demonstrating that gelation could be suppressed for more than a month even at a 6% concentration. In particular, the aqueous solution of the bw753ΔC protein was able to suppress gelation for up to 6 months at a 6% concentration.

[0123] From these results, it has been demonstrated that aqueous solutions of terminal-mutant silk proteins having mutations in the N-terminal and / or C-terminal regions of the silk protein can suppress gelation for a longer period compared to aqueous solutions of wild-type silk protein. All publications, patents and patent applications cited herein are incorporated herein by direct reference.

[0124] 0100...Wild-type silk protein, 0101...N-terminal region, 0102...Central region, 0103...C-terminal region, 0104...Signal peptide, 0105...Repeat unit, 0106...Linking region, 0201...N-terminal region, 0202...Central region, 0203...C-terminal region, 0204...Repeat unit, 0205...Labeled peptide

Claims

1. A terminal region mutant silk protein, wherein the protein is a fibroin protein or a spidoin protein comprising an N-terminal region, a central region, and a C-terminal region in that order from the N-terminus, and the polypeptide comprises a polypeptide having a deletion in either the N-terminal region or the C-terminal region, or / or one or more amino acid mutations of addition, deletion, or substitution in either the N-terminal region or the C-terminal region.

2. The terminal region mutant silk protein according to claim 1, wherein 70% or more of the amino acids constituting the central region consist of glycine residues and alanine residues.

3. The terminal region mutant silk protein according to claim 1 or 2, wherein the fibroin protein is derived from a bagworm or a silkworm.

4. A composition comprising one or more terminal region mutant silk proteins as described in claims 1 to 3.

5. An aqueous solution of silk protein containing one or more terminal region mutant silk proteins as described in claims 1 to 3.

Images