WO2023038154A1

WO2023038154A1 - Large-diameter artificial polypeptide fiber, method for manufacturing same, and adhesive

Info

Publication number: WO2023038154A1
Application number: PCT/JP2022/034270
Authority: WO
Inventors: 徹高橋; 廉伊藤; 聡石原; 聡小池田
Original assignee: Ｓｐｉｂｅｒ株式会社; 天野エンザイム株式会社
Priority date: 2021-09-13
Filing date: 2022-09-13
Publication date: 2023-03-16

Abstract

The present invention provides a method for manufacturing an artificial polypeptide fiber, the method comprising: a step for bundling a plurality of raw material fibers containing an artificial polypeptide; and a step for bringing an obtained bundle of raw material fibers into contact with a composition containing a binder and an enzyme.

Description

Large-diameter artificial polypeptide fiber, method for producing the same, and adhesive

The present invention relates to a large-diameter artificial polypeptide fiber, a method for producing the same, and an adhesive.

Although natural fibers have been used for a long time, it has been difficult to supply large quantities and control fiber forms such as thickness and length. Conventionally, petroleum-derived synthetic fibers have been used to solve these problems. However, due to the recent heightened awareness of environmental conservation, there is a need to break away from petroleum-derived materials, which have a large environmental load, and there is a similar demand in the technical field of textiles.

Therefore, materials that have a small environmental impact and can be mass-produced are attracting attention. For example, Patent Document 1 discloses a biodegradable artificial polypeptide fiber that can be industrially produced in desired thickness and length.

WO2012/165476 JP-A-63-82737

An artificial polypeptide fiber is produced by, for example, extruding a dope solution in which an artificial protein, which is a type of artificial polypeptide, is dissolved in a predetermined solvent to form a linear molded body, and then extruding the linear molded body in a coagulation bath or in the atmosphere. prepared by removing the solvent from However, when attempting to produce a relatively thick artificial polypeptide fiber by a general spinning method, the linear molded product discharged from the spinning nozzle contains a large amount of solvent. It takes time. If the solvent remains in the artificial polypeptide fiber, problems such as the inability to properly form the fiber and the inability to obtain a certain strength occur, resulting in a decrease in the efficiency of fiber production.

When obtaining thick fibers, a method is often used to obtain seemingly thick bundled fibers by bundling thin raw material fibers and adhering them with a synthetic adhesive (for example, Patent Document 2). In Patent Document 2, a large-diameter pile for flocking is manufactured by covering and hardening a plurality of small-diameter piles with a binder. Nitrile rubber, polyurethane rubber, vinyl chloride resin, acrylic acid ester resin, ethylene-vinyl acetate copolymer resin, phenol resin, or epoxy resin is used for the binder. However, the presence of the binder that occupies most of the large-diameter pile can impair the flexibility and biodegradability of the entire fiber.

Therefore, an object of the present invention is to provide an artificial polypeptide fiber having a novel bundle structure and a method for producing the same. Another object of the present invention is to provide an adhesive that can advantageously bond artificial polypeptide fibers.

The present invention provides the following [1] to [17].
[1] A step of bundling a plurality of raw fibers containing an artificial polypeptide;
a step of contacting the obtained bundle of raw material fibers with a composition containing a binder and an enzyme;
A method of making an engineered polypeptide fiber, comprising:
[2] The method for producing an artificial polypeptide fiber according to [1], wherein the enzyme contains an oxidase.
[3] The method for producing an artificial polypeptide fiber according to [2], wherein the oxidase is at least one selected from the group consisting of laccase, bilirubin oxidase, glucose oxidase, and peroxidase.
[4] The method for producing an artificial polypeptide fiber according to any one of [1] to [3], wherein the artificial polypeptide contains a structural protein.
[5] The method for producing an artificial polypeptide fiber according to any one of [1] to [3], wherein the artificial polypeptide contains fibroin.
[6] The method for producing an artificial polypeptide fiber according to any one of [1] to [3], wherein the artificial polypeptide contains spider silk fibroin.
[7] An artificial polypeptide fiber produced by the method according to any one of [1] to [6].
[8] An artificial polypeptide fiber, in which a plurality of raw fibers containing an artificial polypeptide are bonded together via a binder.
[9] The artificial polypeptide fiber of [8], wherein the artificial polypeptide comprises a structural protein.
[10] The artificial polypeptide fiber of [8], wherein the artificial polypeptide contains fibroin.
[11] The artificial polypeptide fiber of [8], wherein the artificial polypeptide comprises spider silk fibroin.
[12] An adhesive for bonding together fibers comprising an artificial polypeptide, comprising:
An adhesive comprising a binder and an enzyme that reacts with at least one of the artificial polypeptide and the binder.
[13] The adhesive of [12], wherein the enzyme is an oxidase.
[14] The adhesive according to [13], wherein the oxidase is at least one selected from the group consisting of laccase, bilirubin oxidase, glucose oxidase, and peroxidase.
[15] The adhesive according to any one of [12] to [14], wherein the artificial polypeptide comprises a structural protein.
[16] The adhesive according to any one of [12] to [14], wherein the artificial polypeptide contains fibroin.
[17] The adhesive according to any one of [12] to [14], wherein the artificial polypeptide comprises spider silk fibroin.

According to the present invention, it is possible to provide an artificial polypeptide fiber having a new bundle-like structure and a method for producing the same by binding a binder and a raw material fiber through an enzymatic reaction. The resulting artificial polypeptide fiber has a bundle-like structure, each of which is directly or indirectly bound by an enzymatic reaction to form a single fiber with a larger diameter. The fibers produced by the method according to the present invention are bound with a binder after producing small-diameter fibers. It is possible to greatly reduce the resistance and increase the manufacturing efficiency.

1 is a schematic diagram showing a cross section of an artificial polypeptide fiber according to one embodiment of the present invention; FIG. FIG. 2 is a schematic diagram showing an example of a domain sequence of artificial fibroin. FIG. 4 is a diagram showing the distribution of z/w (%) values of naturally occurring fibroin. FIG. 4 is a diagram showing the distribution of x/y (%) values of naturally occurring fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of artificial fibroin. 1 is a schematic diagram showing an example of a fibroin domain sequence. FIG. BRIEF DESCRIPTION OF THE DRAWINGS It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing a raw material fiber. 1 is a photograph showing an example of a method for producing an artificial polypeptide according to an embodiment of the present invention;

The present invention will be described in detail below.

[First Embodiment]
A first embodiment of the present invention includes a step of bundling a plurality of raw fibers containing an artificial polypeptide, and a step of contacting the resulting bundle of raw fibers with a composition containing a binder and an enzyme. A method for producing an artificial polypeptide fiber.

According to the method of the present embodiment, by bundling a plurality of raw fibers containing an artificial polypeptide and bringing them into contact with a composition containing a binder and an enzyme, the enzyme binds to at least one of the artificial polypeptide and the binder. By reacting to chemically cross-link at least one of raw material fiber to raw material fiber, raw material fiber to binder, and binder to binder, an artificial polypeptide fiber can be produced.

The step of bundling a plurality of raw fibers (artificial polypeptide fibers with a small diameter) may be a step of simply gathering the raw fibers into a bundle by hand. It may be a process. The cylindrical container is not particularly limited, and any cylindrical container having a desired inner diameter can be used. The inner diameter of the cylindrical container is preferably a size suitable for the thickness of the target artificial polypeptide fiber, for example, 1.2 to 2 times the size of the target artificial polypeptide fiber. , preferably 1.3 to 1.7 times as large. By using a cylindrical container having such a size, it is possible to create a state in which the raw material fibers are closer to each other, and the effect of facilitating cross-linking by an enzymatic reaction can be expected.

The number of raw material fibers to be bundled can be appropriately adjusted in the range of, for example, 5 to 100, 10 to 90, 20 to 80, 30 to 70, or 40 to 60. The number of raw material fibers to be bundled is reflected in the fiber diameter of the artificial polypeptide fiber (large-diameter artificial polypeptide fiber), and the larger the number of raw material fibers, the thicker the resulting artificial polypeptide fiber can be.

[Raw material fiber]
The raw material fiber is a fiber containing an artificial polypeptide, or may be a fiber consisting only of an artificial polypeptide. An engineered polypeptide may include a backbone derived from an engineered protein. Engineered proteins may include engineered structural proteins. Artificial structural proteins may include artificial fibroin. Artificial fibroin may include artificial modified spider silk fibroin.

<Artificial protein>
Examples of artificial proteins include any proteins that can be produced on an industrial scale, such as proteins that can be used for industrial purposes. The term "applicable for industrial use" means, for example, that it can be used for various general-purpose materials used indoors and outdoors, and various materials used for medical applications, clothing applications, and the like. Specific examples of proteins that can be used industrially include structural proteins. Specific examples of structural proteins include spider silk, silkworm silk, keratin, collagen, elastin, resilin, and proteins derived from these. As the protein to be used, artificial fibroin is preferable, and artificial spider silk fibroin (artificial modified spider silk fibroin) is more preferable.

Artificial proteins include recombinant proteins and synthetic proteins. In other words, the term "artificial protein" as used herein means an artificially produced protein. An artificial protein may be a protein whose domain sequence differs from the amino acid sequence of a naturally occurring protein, or may be a protein whose amino acid sequence is identical to that of a naturally occurring protein. In addition, the "artificial protein" may be one that uses the amino acid sequence of a naturally-occurring protein as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of a naturally-occurring protein (for example, a cloned naturally-occurring protein). The amino acid sequence may be modified by modifying the gene sequence of the derived protein), or artificially designed and synthesized without relying on naturally occurring proteins (e.g., those that encode the designed amino acid sequence a desired amino acid sequence obtained by chemically synthesizing a nucleic acid).

The artificial polypeptide may have 50 or more amino acid residues. The number of amino acid residues is, for example, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more. The number of amino acid residues is, for example, 5000 or less, 4500 or less, 4000 or less, 3500 or less, 3000 or less, 2500 or less, 2000 or less, 1500 or less, 1000 or less. Solubility in solvents tends to increase as the number of amino acid residues decreases.

The artificial polypeptide may have a hydrophilic polypeptide backbone or a hydrophobic polypeptide backbone. When the artificial polypeptide has a hydrophobic polypeptide backbone, the properties of the raw material fiber can be shifted to the hydrophobic side compared to when the artificial polypeptide has a hydrophilic polypeptide backbone. It becomes possible to control the hydrophobicity of the entire fiber over a wider range.

The hydrophobicity of the hydrophobic polypeptide backbone can be estimated using the average hydropathic index value described below as an index. The average hydropathic index value of the hydrophobic polypeptide is, for example, 0.00 or more, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more. , 0.40 or greater, 0.45 or greater, 0.50 or greater, 0.55 or greater, 0.60 or greater, 0.65 or greater, or 0.70 or greater. Although the upper limit is not particularly limited, it may be, for example, 1.00 or less, or 0.7 or less.

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

The hydrophobic polypeptide skeleton preferably has a large contact angle with water. The contact angle of water is measured by using a membrane composed of a polypeptide obtained by decomposing a compound (polypeptide) corresponding to the hydrophobic polypeptide skeleton or a synthetic polymer and isolating only the hydrophobic polypeptide skeleton on a substrate. can be formed and evaluated using the film. A polypeptide that forms a film with a contact angle of 55° or more after 5 seconds of water being dropped onto the film is preferred as the hydrophobic polypeptide backbone. The contact angle may be, for example, 60° or greater, 65° or greater, or 70° or greater.

The hydrophobic polypeptide backbone is preferably one with excellent hot water resistance. Hydrothermal resistance can be evaluated using a compound (polypeptide) corresponding to the hydrophobic polypeptide backbone or a polypeptide obtained by decomposing a synthetic polymer and isolating only the hydrophobic polypeptide backbone. A dispersion containing the above-mentioned polypeptide and water, wherein the content of the above-mentioned polypeptide is 5% by mass, is prepared, and the dispersion is not decomposed even if the dispersion is heat-treated at 100°C for 5 hours. Polypeptides are preferred as the hydrophobic polypeptide backbone.

The polypeptide scaffold according to this embodiment may be, for example, a structural protein. A structural protein means a protein involved in the structure of a living body, a protein constituting a structure produced by a living body, or a protein derived from them. Structural proteins also refer to proteins that self-aggregate under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. Furthermore, a structural protein can be said to be a protein in which a characteristic amino acid sequence or a motif consisting of several amino acid residues is repeatedly present and which forms the skeleton of an organism or material. Structural proteins in nature include, for example, fibroin, keratin, collagen, elastin and resilin.

The structural protein may be an artificial structural protein. As used herein, the term "artificial structural protein" means a structural protein that is artificially produced. The artificial structural protein may be a structural protein produced by microorganisms through genetic recombination, and includes those with improved amino acid sequences from the viewpoint of moldability and productivity, and is not limited to the sequence of a naturally occurring structural protein.

When artificially forming a protein with an artificial structure, amino acids with smaller side chains are more likely to form hydrogen bonds, making it easier to obtain a strong molded product. In addition, since alanine and glycine residues are amino acids with non-polar side chains, they are arranged so as to face inward during the folding process during polypeptide production, and tend to adopt an α-helical structure or a β-sheet structure. Therefore, it is desirable that the ratio of amino acids such as glycine residues, alanine residues and serine residues is high. From the viewpoint of obtaining a molded article with better strength, the alanine residue content may be, for example, 10 to 40%, 12 to 40%, 15 to 40%, 18 to 40%, 20 to 40%, or It may be 22-40%. From the viewpoint of obtaining a molded article with better strength, the glycine residue content may be, for example, 10 to 55%, 11 to 55%, 13 to 55%, 15 to 55%, 18 to 55%, 20 to 55%. It may be 55%, 22-55%, or 25-55%.

As used herein, "alanine residue content" is a value represented by the following formula.

Alanine residue content = (number of alanine residues contained in polypeptide/number of total amino acid residues in polypeptide) x 100 (%)
In addition, the glycine residue content, serine residue content, threonine residue content, proline residue content and tyrosine residue content are defined in the above formulas, in which alanine residues are replaced with glycine residues, serine residues, It is synonymous with what is read as threonine residue, proline residue and tyrosine residue.

An artificial structural protein according to one embodiment may have a repeat sequence. That is, the polypeptide according to this embodiment may have a plurality of amino acid sequences (repetitive sequence units) with high sequence identity within the polypeptide. The number of amino acid residues in the repeat sequence unit is preferably 6-200. Also, the sequence identity between repeat sequence units may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In addition, the hydrophobicity (hydropathic index) of the repeating sequence unit is, for example, -0.80 or more, -0.70 or more, -0.60 or more, -0.50 or more, -0.40 or more, - 0.30 or more, -0.20 or more, -0.10 or more, 0.00 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more. Although the upper limit of the hydrophobicity of the repeating sequence unit is not particularly limited, it may be, for example, 1.0 or less, or 0.7 or less.

An artificial structural protein according to one embodiment may comprise an (A) _n motif. As used herein, the (A) _n motif means an amino acid sequence mainly composed of alanine residues. (A) The number of amino acid residues in _{the n} motif may be 2-27 and may be an integer from 2-20, 2-16, or 2-12. In addition, (A) the ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).

Artificial fibroin is preferable as the artificial structural protein. Fibroin includes, for example, naturally occurring fibroin. Naturally occurring fibroin includes, for example, fibroin produced by insects or arachnids.

Examples of fibroin produced by insects include, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, and Pilos cylinthia silkworm. ), silk proteins produced by silkworms such as Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, and exhaled by Vespa simillima xantoptera larvae Hornet silk protein.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (nucleotide sequence), AAA27840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include, for example, spiders belonging to the genus Araneus such as Araneus spiders, Araneus spiders, Red spiders, Green spiders, and Beetle spiders; Spiders belonging to the genus Pronus, spiders belonging to the genus Pronus such as spiders belonging to the Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius such as the genus Ordgarius and the spiders belonging to the genus Ordgarius, spiders belonging to the genus Argiope such as the argiope spider, the argiope spider, and the argiope spider, and the like Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas, such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, and spiders Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as spiders belonging to the genus Cyclosa, spiders belonging to ), spiders belonging to the genus Cyclosa, and spiders belonging to the genus Chorizopes, such as spiders and spiders, Spiders belonging to the genus Tetragnatha such as the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucauge, such as the genus Leucauge, and the genus Nephila Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as golden spiders, spiders belonging to the genus Dyschiriognatha such as the brown spider, widow spiders such as the black widow spider, the redback spider, the gray widow spider and the western widow spider Spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Latrodectus and spiders belonging to the genus Euprosthenops, produce Spider silk proteins are included. Spider silk proteins include, for example, dragline proteins such as MaSp (MaSp1 and MaSp2), ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

More specific examples of fibroin produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin- 4(adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major angu11ate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila accession number 2Lan4Aclavata] (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Eanprosthenops] Accession number CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor amplified silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor amplified silk protein 2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), and minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank Accession No. ABR37278.1 (amino acid sequence)).

A more specific example of naturally-derived fibroin is fibroin whose sequence information is registered in NCBI GenBank. For example, among the sequence information registered in NCBI GenBank, among the sequences that include INV as a division, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION It can be confirmed by extracting the specific product character string from the sequence, CDS, and the specific character string from SOURCE to TISSUE TYPE.

As used herein, "artificial fibroin" means artificially produced fibroin (artificial fibroin). Artificial fibroin may be fibroin that differs from the amino acid sequence of naturally-occurring fibroin, or fibroin that is identical to the amino acid sequence of naturally-occurring fibroin.

The artificial fibroin may be a fibrous protein having a structure similar to that of naturally occurring fibroin, or may be fibroin having a sequence similar to the repeating sequence of naturally occurring fibroin. The “sequence similar to the repeat sequence possessed by fibroin” may be a sequence actually possessed by naturally occurring fibroin, or may be a sequence similar thereto.

"Artificial fibroin" refers to those obtained by modifying the amino acid sequence of naturally-occurring fibroin (e.g., modifying the gene sequence of cloned naturally-occurring fibroin), as long as it has the amino acid sequence specified in the present disclosure. The amino acid sequence may be modified by the method), or the amino acid sequence may be artificially designed without relying on naturally occurring fibroin (for example, by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). having a desired amino acid sequence). An artificial fibroin with a modified amino acid sequence is also included in the artificial fibroin as long as the amino acid sequence differs from the amino acid sequence of the naturally-derived fibroin. Examples of artificial fibroin include artificial silk fibroin (modified amino acid sequence of silk protein produced by silkworms), and artificial spider silk fibroin (modified amino acid sequence of spider silk protein produced by spiders). ) and the like. When the synthetic polymer according to the present disclosure is used as a molding material, spider silk fibroin is relatively easy to fibrillate and has high fiber-forming ability, so the artificial fibroin preferably contains artificial spider silk fibroin, more preferably Consists of artificial spider silk fibroin.

As a specific example of the artificial fibroin according to the present embodiment, an artificial fibroin (first artificial fibroin) derived from a dragline silk protein produced in the major pituitary gland of spiders, a glycine residue content of artificial fibroin with reduced domain sequence (second artificial fibroin), (A) artificial fibroin with domain sequence with reduced _n- motif content (third artificial fibroin), content of glycine residues, and (A) an artificial fibroin with a reduced _n- motif content (fourth artificial fibroin), an artificial fibroin having a domain sequence containing a region with a locally large hydrophobicity index (fifth artificial fibroin), and glutamine Artificial fibroins with domain sequences with reduced content of residues (sixth artificial fibroin) are included.

A first artificial fibroin includes a protein comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In the first artificial fibroin, the number of amino acid residues in the (A) _n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, and an integer of 10 to 20. is even more preferred, integers from 4 to 16 are even more preferred, integers from 8 to 16 are particularly preferred, and integers from 10 to 16 are most preferred. In the first artificial fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. and even more preferably 20 to 75 residues. In the first artificial fibroin, the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m is amino acid residue It is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more of the total number.

The first artificial fibroin comprises an amino acid sequence unit represented by Formula 1: [(A) _n motif-REP] _m and has an amino acid sequence whose C-terminal sequence is shown in any one of SEQ ID NOS: 1 to 3, or The polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the C-terminal 50 amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 has the sequence It is identical to the amino acid sequence shown in No. 1 with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID No. 3 has 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID No. 1. Identical to the amino acid sequence.

As a more specific example of the first artificial fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3 KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 and 90 Artificial fibroin comprising amino acid sequences having greater than % sequence identity can be mentioned. Preferably, the sequence identity is 95% or greater.

The amino acid sequence shown by SEQ ID NO: 4 is the amino acid sequence (SEQ ID NO: 5) consisting of an initiation codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus of ADF3. The 13th repeat region was increased to approximately double and mutated so that translation stops at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown by SEQ ID NO:4 is identical to the amino acid sequence shown by SEQ ID NO:3.

The (1-i) artificial fibroin may consist of the amino acid sequence shown in SEQ ID NO:4.

The second artificial fibroin has an amino acid sequence in which the domain sequence has a reduced content of glycine residues compared to the naturally-derived fibroin. The second artificial fibroin can be said to have an amino acid sequence corresponding to at least one or more glycine residues in REP being replaced with another amino acid residue, as compared with the naturally occurring fibroin. .

The second artificial fibroin has a domain sequence of GGX and GPGXX (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine) in REP compared to the naturally-derived fibroin. In at least one motif sequence selected from ), it has an amino acid sequence corresponding to at least one or more glycine residues in the motif sequence being replaced with another amino acid residue may

In the second artificial fibroin, the percentage of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.

The second artificial fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the domain sequence to the (A) _n motif located on the most C-terminal side to the domain sequence Let z be the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence When w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence, z/w is 30% or more, It may have an amino acid sequence that is 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

The second artificial fibroin preferably has an increased content of the amino acid sequence consisting of XGX by substituting one glycine residue in the GGX motif with another amino acid residue. 6. In the second artificial fibroin, the content of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, even more preferably 10% or less. % or less, even more preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z/w) of the amino acid sequence consisting of XGX below.

A method for calculating z/w will be described in more detail. First, in a fibroin (artificial fibroin or naturally occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , the (A) _n located on the most C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50×3=150. In addition, for example, when there is an X (central X) contained in two XGX, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues be). w is the total number of amino acid residues contained in the domain sequence, excluding the sequence from the (A) _n motif positioned most on the C-terminal side to the C-terminus of the domain sequence. For example, for the domain sequence shown in Figure 3, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the most C-terminal (A) _n motif). Then z/w (%) can be calculated by dividing z by w.

Here, z/w in naturally occurring fibroin will be explained. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (among them, 415 types of arachnid-derived fibroin) were extracted. Naturally derived fibroin containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m and having an amino acid sequence consisting of GGX in fibroin content of 6% or less among all the extracted fibroin z/w was calculated from the amino acid sequence of fibroin in the above-described calculation method. The results are shown in FIG. The horizontal axis of FIG. 3 indicates z/w (%), and the vertical axis indicates frequency. As is clear from FIG. 3, the z/w for naturally occurring fibroin is all less than 50.9% (50.86% being the highest).

In the second artificial fibroin, z/w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, and 70% or more. and even more preferably 80% or more. Although the upper limit of z/w is not particularly limited, it may be, for example, 95% or less.

The second artificial fibroin can be obtained, for example, by substituting at least part of the nucleotide sequence encoding the glycine residue from the cloned naturally-derived fibroin gene sequence so as to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z/w is 50.9% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the alteration corresponding to replacing the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , insertions and/or additions may be made to the amino acid sequence.

The other amino acid residue mentioned above is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( Hydrophobic amino acid residues such as M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W) residues, glutamine (Q) residues, asparagine (N) residues, serine (S ) residues, lysine (K) residues and glutamic acid (E) residues are preferred, and hydrophilic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

More specific examples of the second artificial fibroin include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (2-ii) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of artificial fibroin.

The artificial fibroin of (2-i) will be explained. The amino acid sequence shown by SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the amino acid sequence shown by SEQ ID NO: 6 from the N-terminal side to the C-terminal side, and furthermore, in front of the C-terminal sequence. [(A) _n motif-REP] is inserted into the . The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the C-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and a His tag sequence are added to the C-terminus of a sequence in which is repeated four times.

The z/w value in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z/w values in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are each 58.7%, 70.1%, 66.1% and 70.0%. In addition, the value of x/y in the jagged ratio (described later) of 1:1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 15.0%, 15.0%, 93.4%, 92.7% and 89.8% respectively.

The (2-i) artificial fibroin may consist of the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The artificial fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The artificial fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-ii) artificial fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and XGX contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

The second artificial fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This makes it possible to isolate, immobilize, detect and visualize artificial fibroin.

Examples of tag sequences include affinity tags that utilize specific affinity (binding, affinity) with other molecules. A specific example of the affinity tag is a histidine tag (His tag). A His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and because it has the property of specifically binding to metal ions such as nickel, artificial fibroin is isolated by metal chelating chromatography. can be used for A specific example of the tag sequence is the amino acid sequence represented by SEQ ID NO: 11 (an amino acid sequence containing a His tag sequence and a hinge sequence).

In addition, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.

Furthermore, it is also possible to use "epitope tags" that utilize antigen-antibody reactions. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag, and the like. Artificial fibroin can be easily purified with high specificity by using an epitope tag.

Furthermore, a tag sequence that can be cleaved by a specific protease can also be used. Artificial fibroin from which the tag sequence has been cut off can also be recovered by treating the protein adsorbed via the tag sequence with protease.

As more specific examples of artificial fibroin containing a tag sequence, (2-iii) the amino acid represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

The amino acid sequences shown in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in SEQ ID NO: 11 (including His tag sequence and hinge sequence) was added to the N-terminus of the amino acid sequence shown.

The (2-iii) artificial fibroin may consist of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The (2-iv) artificial fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The artificial fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-iv) artificial fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

The second artificial fibroin may contain a secretion signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A third artificial fibroin has an amino acid sequence in which the domain sequence has a reduced content of (A) _n motifs compared to the naturally-occurring fibroin. The domain sequence of the third artificial fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or a plurality of (A) _n motifs compared to the naturally-occurring fibroin.

The third artificial fibroin may have an amino acid sequence corresponding to 10-40% deletion of the (A) _n motif from the naturally-occurring fibroin.

The third artificial fibroin has a domain sequence at least one (A) _n motif for every 1-3 (A) _n motifs from the N-terminal side to the C-terminal side compared to the naturally occurring fibroin. may have an amino acid sequence corresponding to the deletion of

The third artificial fibroin has at least two consecutive (A) _n motif deletions from the N-terminal side to the C-terminal side and one (A ) may have an amino acid sequence corresponding to deletion of _n motifs repeated in this order.

The third artificial fibroin may have an amino acid sequence corresponding to deletion of at least every two (A) _n motifs from the N-terminal side to the C-terminal side of the domain sequence. .

The third artificial fibroin comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , with two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -REP] unit sequentially compares the number of amino acid residues of REP, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11. When the maximum value of the total sum of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units equal to 3 is x, and the total number of amino acid residues of the domain sequence is y Furthermore, it may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

A method for calculating x/y will be described in more detail with reference to FIG. FIG. 2 shows the domain sequence of the artificial fibroin with the N-terminal and C-terminal sequences removed. The domain sequence is, from the N-terminal side (left side), (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called _n motif.

Two adjacent [(A) _n- motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, unselected [(A) _n motif-REP] units may be present. In FIG. 2, pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), pattern 4 (comparison of the first REP and A second REP comparison) was shown. Note that there are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two adjacent [(A) _n motif-REP] units selected is compared. Comparison is carried out by determining the ratio of the number of amino acid residues to the number of amino acid residues of the other. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the second REP is The ratio of amino acid residue numbers is 100/50=2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the fifth REP is 30/20=1.5.

In FIG. 2, a set of [(A) _n motif-REP] units having a ratio of 1.8 to 11.3 for the number of amino acid residues of the other is set to 1 for the one with the smaller number of amino acid residues. It is indicated by a solid line. This ratio is referred to herein as the serration ratio. A set of [(A) _n motif-REP] units in which the other amino acid residue number ratio is less than 1.8 or greater than 11.3 when the number of amino acid residues is less than 1 is indicated by a dashed line. Indicated.

In each pattern, the numbers of all amino acid residues of two adjacent [(A) _n motif-REP] units indicated by solid lines are summed up (not only REP but also the number of amino acid residues of (A) _n motifs). be.). Then, the added total values are compared, and the total value (maximum total value) of the pattern with the maximum total value is defined as x. In the example shown in FIG. 2, the total value of pattern 1 is the largest.

Next, x/y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the third artificial fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, and even more preferably 70% or more. Preferably, 75% or more is even more preferable, and 80% or more is particularly preferable. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x /y is preferably 77.1% or more, and when the serration ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the serration ratio is 1 : 1.9 to 4.1, x/y is preferably 64.2% or more.

When the third artificial fibroin is an artificial fibroin in which at least 7 of (A) _n motifs present in the domain sequence are composed only of alanine residues, x/y is 46.4% or more. is preferably 50% or more, more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and 80% or more It is particularly preferred to have The upper limit of x/y is not particularly limited as long as it is 100% or less.

Here, x/y in naturally occurring fibroin will be explained. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (among them, 415 types of arachnid-derived fibroin) were extracted. Of all the extracted fibroin, from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , x/y was calculated. FIG. 4 shows the results when the serration ratio is 1:1.9 to 4.1.

The horizontal axis in FIG. 4 indicates x/y (%), and the vertical axis indicates frequency. As is clear from FIG. 4, the x/y ratios for naturally occurring fibroin are all less than 64.2% (64.14% being the highest).

For the third artificial fibroin, for example, one or more sequences encoding (A) _n motifs are deleted from the cloned natural fibroin gene sequence such that x/y is 64.2% or more. can be obtained by Alternatively, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed such that x/y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence described above. In any case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are substituted, deleted, inserted and/or added. Alterations in the amino acid sequence corresponding to what has been done may be made.

More specific examples of the third artificial fibroin include (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of artificial fibroin.

The artificial fibroin of (3-i) will be explained. The amino acid sequence represented by SEQ ID NO: 17 is every two (A) _n The motif was deleted and one [(A) _n motif-REP] was inserted in front of the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described for the second artificial fibroin.

The value of x/y at the jagged ratio of 1:1.8 to 11.3 of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 15.0%. The x/y values of the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%. The z/w values in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.0%, respectively. 1% and 70.0%.

The (3-i) artificial fibroin may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The artificial fibroin (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The artificial fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The artificial fibroin of (3-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from the N-terminal side to the C-terminal side , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (Giza ratio is 1:1.8 to 11.3) It is preferable that x/y is 64.2% or more, where x is the maximum sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.

The third artificial fibroin may contain the tag sequence described above at either or both of the N-terminus and C-terminus.

As more specific examples of artificial fibroin containing a tag sequence, (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) sequence, or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 have SEQ ID NO: 11 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in (including His tag sequence and hinge sequence) is added.

The artificial fibroin of (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The (3-iv) artificial fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The artificial fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(3-iv) artificial fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Let x be the maximum value of the total sum of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. , x/y is preferably 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

The third artificial fibroin may contain a secretion signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The fourth artificial fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motifs and a reduced content of glycine residues compared to naturally occurring fibroin. have. The domain sequence of the fourth artificial fibroin has at least one or more (A) _n motifs deleted, and at least one or more glycine residues in REP is deleted compared to the naturally-derived fibroin. It can be said to have an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth artificial fibroin is an artificial fibroin having both the features of the second artificial fibroin and the third artificial fibroin. Specific aspects and the like are as described in the second artificial fibroin and the third artificial fibroin.

As more specific examples of the fourth artificial fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410 ), the amino acid sequence shown in SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 An artificial fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in . Specific embodiments of the artificial fibroin comprising the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

The fifth artificial fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues with a larger hydrophobicity index than in naturally occurring fibroin, and/or REP It may have an amino acid sequence that includes regions of high local hydrophobicity index corresponding to the insertion of one or more amino acid residues with high hydrophobicity index therein.

A region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

The amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). A residue is more preferred.

The fifth artificial fibroin has one or more amino acid residues in REP substituted with amino acid residues having a larger hydrophobicity index than the naturally occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, one or more amino acid residues are substituted, deleted, inserted and / or added as compared to naturally occurring fibroin There may be alterations in the amino acid sequence corresponding to what has been done.

For the fifth artificial fibroin, for example, one or more hydrophilic amino acid residues (eg, amino acid residues with a negative hydrophobicity index) in REP are replaced with hydrophobic amino acid residues from the cloned natural fibroin gene sequence. by substituting a group (for example, an amino acid residue with a positive hydrophobicity index) and/or by inserting one or more hydrophobic amino acid residues into REP. Further, for example, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acids in REP In addition to modifications corresponding to residue insertions, amino acid sequence modifications corresponding to substitutions, deletions, insertions and/or additions of one or more amino acid residues may also be made.

The fifth artificial fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence Let p be the total number of amino acid residues contained in a region where the average value of the hydrophobic index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequences excluding the sequences from the domain sequence, p/q is 6, where q is the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. It may have an amino acid sequence that is greater than or equal to .2%.

Regarding the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Specifically, the hydropathic index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

A method for calculating p/q will be described in more detail. For the calculation, the sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence (hereinafter referred to as “sequence A”) is used. First, in all REPs contained in sequence A, the average value of the hydrophobic index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to multiple "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

For example, if 20 “continuous 4 amino acid residues with an average hydrophobic index of 2.6 or more” are extracted (no overlap), the average hydrophobic index of 4 consecutive amino acid residues is 2 A region that is 0.6 or greater will contain 20 consecutive 4-amino acid residues (no overlap), and p is 20×4=80. Further, for example, when two "consecutive 4 amino acid residues having an average hydrophobicity index of 2.6 or more" exist overlapping by one amino acid residue, the hydrophobic index of the consecutive 4 amino acid residues A region with an average of 2.6 or more contains 7 amino acid residues (p=2×4−1=7, where “−1” is duplicate subtraction). For example, in the case of the domain sequence shown in FIG. 5, there are seven non-overlapping “continuous 4-amino acid residues with an average hydrophobicity index of 2.6 or more”, so p is 7×4= 28. For example, in the case of the domain sequence shown in FIG. 5, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A) _n motif present at the end of the C-terminal side). Then p/q (%) can be calculated by dividing p by q. In the case of FIG. 5, 28/170=16.47%.

In the fifth artificial fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, and 20% or more. Even more preferably, it is still more preferably 30% or more. Although the upper limit of p/q is not particularly limited, it may be, for example, 45% or less.

The fifth artificial fibroin is, for example, the amino acid sequence of the cloned naturally-occurring fibroin so as to satisfy the above p/q conditions, and one or more hydrophilic amino acid residues in REP (for example, a hydrophobicity index). negative amino acid residue) with a hydrophobic amino acid residue (e.g., an amino acid residue with a positive hydrophobicity index), and/or inserting one or more hydrophobic amino acid residues in REP can be obtained by locally modifying an amino acid sequence containing a region with a large hydrophobicity index. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above p/q conditions from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more amino acid residues in REP have been replaced with amino acid residues having a higher hydrophobicity index compared to naturally-occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, further modification corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues may be performed. .

Amino acid residues with a large hydrophobicity index are not particularly limited, but areoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.

As more specific examples of the fifth artificial fibroin, (5-i) an amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666); or (5-ii) artificial fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

(5-i) artificial fibroin will be explained. The amino acid sequence shown by SEQ ID NO: 19 consists of 3 amino acid residues every other REP except for the terminal domain sequence on the C-terminal side with respect to the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410). The amino acid sequence (VLI) was inserted at two sites, some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids on the C-terminal side were deleted. The amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) in which an amino acid sequence (VLI) consisting of three amino acid residues is inserted at every other REP. be. The amino acid sequence shown by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 8.

The (5-i) artificial fibroin may consist of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The artificial fibroin (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The artificial fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The artificial fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

The fifth artificial fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus.

More specific examples of artificial fibroin containing a tag sequence include (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv ) Artificial fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are added to the N-terminals of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, where the amino acid sequence shown in SEQ ID NO: 11 (His tag sequence and hinge sequence) are added.

The artificial fibroin of (5-iii) may consist of the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The (5-iv) artificial fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. The artificial fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(5-iv) artificial fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

The fifth artificial fibroin may contain a secretion signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A sixth artificial fibroin has an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring fibroin.

The sixth artificial fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the REP amino acid sequence.

When the sixth artificial fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.

As used herein, the "GPGXX motif content" is a value calculated by the following method.

Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (artificial fibroin or naturally occurring fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total by three (that is, the total number of G and P in the GPGXX motif) is defined as s, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is taken from the domain sequence. The GPGXX motif content rate is calculated as s/t, where t is the total number of amino acid residues in all REPs excluding (A) _n motifs.

In the calculation of the GPGXX motif content rate, the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is "the most C-terminal side (A) The sequence from _{the n} motif to the C-terminus of the domain sequence” (the sequence corresponding to REP) may include sequences that are poorly correlated with sequences characteristic of fibroin, and m is small If the domain sequence is short (that is, if the domain sequence is short), it affects the calculation result of the GPGXX motif content rate, so this effect is to be eliminated. When a "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA".

FIG. 2 is a schematic diagram showing the domain sequence of artificial fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the artificial fibroin domain sequence (“[(A) _n motif-REP] _m -(A) _n motif” type) shown in FIG. (A) The sequence obtained by removing the sequence from _{the n} motif to the C-terminus of the domain sequence from the domain sequence” (sequence shown as “region A” in FIG. 2). The number of GPGXX motifs is 7, and s is 7×3=21. Similarly, all REPs are "sequences obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" (the sequence shown as "region A" in FIG. .), the total number of amino acid residues t of all REPs further excluding the (A) _n motif from the sequence is 50+40+10+20+30=150. Next, s/t (%) can be calculated by dividing s by t, which is 21/150=14.0% for artificial fibroin in FIG.

The sixth artificial fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%. .

As used herein, the "glutamine residue content" is a value calculated by the following method.

Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (artificial fibroin or naturally occurring fibroin), all contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence (sequence corresponding to "region A" in FIG. 2) REP, the total number of glutamine residues contained in the region is u, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and (A) _n The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues in all REPs excluding motifs. In the calculation of the glutamine residue content, the target is the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because of the reasons described above. It is the same.

The sixth artificial fibroin has a domain sequence corresponding to deletion of one or more glutamine residues in REP or substitution with other amino acid residues as compared to the naturally-occurring fibroin. It may have an amino acid sequence.

"Another amino acid residue" may be an amino acid residue other than a glutamine residue, but preferably an amino acid residue with a higher hydrophobicity index than a glutamine residue. Hydrophobicity indexes of amino acid residues are shown in Table 1.

As shown in Table 1, amino acid residues having a higher hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H); can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

The sixth artificial fibroin has a REP hydrophobicity (hydropathy index) of, for example, -0.80 or more, -0.70, -0.60 or more, -0.50 or more, or -0.40 or more. , -0.30 or more, -0.20 or more, -0.10 or more, 0.00 or more, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0 .35 or greater, 0.40 or greater, 0.45 or greater, 0.50 or greater, 0.55 or greater, 0.60 or greater, 0.65 or greater, or 0.70 or greater. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or less, or 0.7 or less.

"REP hydrophobicity" as used herein is a value calculated by the following method.

Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (artificial fibroin or naturally occurring fibroin), all contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence (sequence corresponding to "region A" in FIG. 2) In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is v, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues in all REPs excluding _n motifs. In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

A sixth artificial fibroin has a domain sequence lacking one or more glutamine residues in REP and/or one or more glutamine residues in REP compared to the naturally-occurring fibroin In addition to the modification corresponding to the substitution of another amino acid residue, there may be modifications of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. .

The sixth artificial fibroin is produced, for example, by deleting one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence, and/or by deleting one or more glutamine residues in REP. can be obtained by substituting the amino acid residue of Also, for example, deletion of one or more glutamine residues in REP from the amino acid sequence of naturally occurring fibroin, and/or substitution of one or more glutamine residues in REP with other amino acid residues It can also be obtained by designing an amino acid sequence corresponding to , and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

More specific examples of the sixth artificial fibroin include (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or sequence artificial fibroin comprising the amino acid sequence represented by number 42 (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42, and an artificial fibroin comprising an amino acid sequence having 90% or more sequence identity.

The artificial fibroin in (6-i) will be explained. The amino acid sequence shown by SEQ ID NO:25 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 (Met-PRT410) with VL. The amino acid sequence shown by SEQ ID NO: 26 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with TS, and replacing the remaining Q with A. The amino acid sequence shown by SEQ ID NO: 27 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VL, and replacing the remaining Q with I. The amino acid sequence shown by SEQ ID NO:28 is obtained by substituting VI for all QQs in the amino acid sequence shown by SEQ ID NO:7 and L for the remaining Qs. The amino acid sequence shown by SEQ ID NO: 29 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VF, and replacing the remaining Q with I.

The amino acid sequence shown by SEQ ID NO: 30 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence shown by SEQ ID NO: 31 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 8 with VL, and replacing the remaining Q with I.

The amino acid sequence represented by SEQ ID NO: 32 consists of 20 domain sequence regions present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) that are repeated twice, and all QQ in the sequence are replaced with VF, And the remaining Q is replaced with I.

The amino acid sequence shown by SEQ ID NO: 41 (Met-PRT917) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with LI and the remaining Qs with V. The amino acid sequence shown by SEQ ID NO:42 (Met-PRT1028) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 with IF, and replacing the remaining Q with T.

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all glutamine residues The group content is below 9% (Table 2).

The artificial fibroin of (6-i) is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It may consist of the amino acid sequence shown.

The artificial fibroin of (6-ii) is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The artificial fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

The artificial fibroin (6-ii) preferably has a glutamine residue content of 9% or less. In addition, the artificial fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.

The sixth artificial fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This makes it possible to isolate, immobilize, detect and visualize artificial fibroin.

More specific examples of artificial fibroin containing a tag sequence include (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028) artificial fibroin comprising an amino acid sequence, or ( 6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 and 90 Artificial fibroins comprising amino acid sequences with greater than % sequence identity can be mentioned.

The amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are SEQ ID NO: 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 at the N-terminus of SEQ ID NO: 11 The amino acid sequence (including the His-tag sequence and hinge sequence) is added. Since the tag sequence was only added to the N-terminus, there was no change in the glutamine residue content, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

The artificial fibroin of (6-iii) is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It may consist of the amino acid sequence shown.

(6-iv) artificial fibroin is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The artificial fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

The artificial fibroin (6-iv) preferably has a glutamine residue content of 9% or less. In addition, the artificial fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

The sixth artificial fibroin may contain a secretion signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

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

The artificial fibroin according to this embodiment also has a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif It may be a protein containing The artificial fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeated amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically corresponding to the REP of the amino acid sequence). ) and is represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif means an array. Here, the (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues is 80% or more of the total number of amino acid residues in the (A) _n motif. REP indicates an amino acid sequence composed of 10-200 amino acid residues. m represents an integer of 10-300. m is preferably an integer of 20-300, more preferably an integer of 30-300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

At least seven of the (A) _n motifs present in the domain sequence are preferably composed only of alanine residues. Consisting only of alanine residues means that the (A) _n motif has an amino acid sequence represented by (Ala) _k (Ala represents an alanine residue). k is preferably an integer of 4-27, more preferably an integer of 4-20, and still more preferably an integer of 4-16.

REP is composed of 10 to 200 amino acid residues. One or more of the amino acid residues constituting REP may be amino acid residues selected from the group consisting of glycine residues, serine residues, and alanine residues. That is, REP may contain amino acid residues selected from the group consisting of glycine residues, serine residues, and alanine residues.

One or more of the amino acid residues constituting REP may be hydrophobic amino acid residues. That is, REP preferably contains hydrophobic amino acid residues. A hydrophobic amino acid residue means an amino acid residue with a positive hydrophobicity index. Regarding the hydrophobicity index of amino acid residues (hydropathy index, hereinafter also referred to as "HI"), a known index known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Hydrophobic amino acid residues include, for example, isoleucine (HI: 4.5), valine (HI: 4.2), leucine (HI: 3.8), phenylalanine (HI: 2.8), methionine (HI: 1.9) and alanine (HI: 1.8).

In the artificial fibroin according to this embodiment, the domain sequence preferably has an amino acid sequence corresponding to the insertion of a cysteine residue into REP, as compared with naturally occurring fibroin.

The domain sequence preferably has an amino acid sequence corresponding to the insertion of a cysteine residue at a position adjacent to a glycine residue, a serine residue, or an alanine residue in REP. More preferably, it has an amino acid sequence corresponding to a cysteine residue inserted at a position adjacent to the glycine residue. A cysteine residue in REP may be located between a glycine residue, a serine residue, or an alanine residue and a glycine residue, a serine residue, or an alanine residue; residues.

The domain sequence preferably has an amino acid sequence corresponding to the insertion of a cysteine residue at the position adjacent to the hydrophobic amino acid residue in REP. In this case, hydrophobic amino acid residues are fixed between molecules by hydrophobic interaction. A cysteine residue in REP may be positioned next to a hydrophobic amino acid residue, or may be positioned between a hydrophobic amino acid residue and an amino acid residue other than a hydrophobic amino acid residue. , a hydrophobic amino acid residue and a glycine residue, a serine residue, or an alanine residue, and may be positioned between a hydrophobic amino acid residue and a glycine residue. Hydrophobic amino acid residues may be one selected from the group consisting of isoleucine residues, valine residues, leucine residues, phenylalanine residues, methionine residues, and alanine residues.

The domain sequence has an amino acid sequence corresponding to the insertion of a cysteine residue into the REP located near the N-terminus and/or C-terminus of the domain sequence compared to naturally-occurring fibroin. good. In this case, the molecular chain can be lengthened. As used herein, a REP located near the N-terminus of a domain sequence means a REP located 1st to 3rd from the N-terminus of the domain sequence. For example, a cysteine residue may be located in the REP positioned 1-2 from the N-terminus of the domain sequence. As used herein, a REP located near the C-terminus of a domain sequence means a REP located 1st to 3rd from the C-terminus of the domain sequence. For example, a cysteine residue may be located in the REP positioned 1-2 from the C-terminus of the domain sequence. Cysteine residues are preferably located in the most N-terminal and/or most C-terminal REP of the domain sequence.

The domain sequence may have an amino acid sequence corresponding to the insertion of a cysteine residue at or near the center in REP compared to naturally occurring fibroin. As used herein, the vicinity of the center of the amino acid sequence in REP refers to the amino acid residue located in the center of REP (when there are two amino acid residues located in the center, the amino acid residue on the N-terminal side) to N 1st to 5th positions toward the terminal side, or 1st to 5th amino acid residues located in the center of REP (when there are two amino acid residues located in the center, amino acid residues on the C-terminal side) indicates the position of For example, the cysteine residue may be located in the center of REP, and is located at the 1st to 3rd or 1st to 2nd positions toward the N-terminal side or the C-terminal side from the amino acid residue located in the center of REP. You may have

The artificial fibroin preferably contains a GPGXX motif (G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than a glycine residue) in the amino acid sequence of REP. Inclusion of this motif in REP can improve the elongation of the artificial fibroin.

When the artificial fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. In this case, the stress of the artificial fibroin fiber becomes even higher. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.

As used herein, the "GPGXX motif content" is a value calculated by the following method. Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif, the most C-terminal In all REPs contained in the sequence excluding the sequence from the located (A) _n motif to the C-terminus of the domain sequence from the domain sequence, triple the total number of GPGXX motifs contained in that region (i.e., (corresponding to the total number of G and P in the GPGXX motif) is c, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and the (A) _n motif is further removed. The GPGXX motif content is calculated as c/d, where d is the total number of amino acid residues in all REPs removed.

FIG. 6 is a schematic diagram showing the domain sequence of fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the fibroin domain sequence (“[(A) _n motif-REP] _m -(A) _n motif” type) shown in FIG. A) GPGXX for calculating c because it is included in the sequence obtained by removing the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence (sequence shown as “region A” in FIG. 6) The number of motifs is 7, and c is 7×3=21. Similarly, all REPs are "sequences obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" (the sequence shown as "region A" in FIG. .), the total number of amino acid residues d of all REPs excluding the (A) _n motif from the sequence is 50+40+10+20+30=150. Next, c/d (%) can be calculated by dividing c by d, which is 21/150=14.0% for fibroin in FIG.

Artificial fibroin has a REP hydrophobicity (hydropathy index: hydrophobicity index) of, for example, -0.80, -0.70, -0.06 or more, -0.50 or more, -0.40 or more. , -0.30 or more, -0.20 or more, -0.10 or more, 0.00 or more, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0 0.35 or greater, 0.40 or greater, 0.45 or greater, 0.50 or greater, 0.55 or greater, 0.60 or greater, 0.65 or greater, or 0.70 or greater. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or less, or 0.7 or less.

As used herein, the "hydrophobicity of REP" is a value calculated by the following method. Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif, the most C-terminal In all REPs contained in the sequence obtained by excluding the sequence from the located (A) _n motif to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to “region A” in FIG. 6), each amino acid in the region The sum of the hydrophobicity indices of the residues is e, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and all REPs excluding the (A) _n motif The hydrophobicity of REP is calculated as e/f, where f is the total number of amino acid residues in . In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

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

The artificial fibroin (i) may have only the amino acid sequence shown in SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51. The artificial fibroin of (ii) may have only the amino acid sequence shown in SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55.

The artificial fibroin described above may contain a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection, visualization, etc. of artificial fibroin.

Examples of tag sequences include affinity tags that utilize specific affinity (binding, affinity) with other molecules. A specific example of the affinity tag is a histidine tag (His tag). A His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and because it has the property of specifically binding to metal ions such as nickel, artificial fibroin is isolated by metal chelating chromatography. can be used for A specific example of the tag sequence is the amino acid sequence represented by SEQ ID NO: 70 or SEQ ID NO: 71 (amino acid sequence containing a His tag).

More specific examples of artificial fibroin containing a tag sequence include (iii) SEQ ID NO: 60 (PRT15), SEQ ID NO: 61 (PRT16), SEQ ID NO: 62 (PRT17), SEQ ID NO: 63 (PRT18), SEQ ID NO: 64 (PRT19) ), or an artificial fibroin comprising the amino acid sequence shown in SEQ ID NO: 65 (PRT20), or (iv) SEQ ID NO: 66 (PRT21), SEQ ID NO: 67 (PRT22), SEQ ID NO: 68 (PRT23), or SEQ ID NO: 69 ( Artificial fibroin containing the amino acid sequence shown in PRT24) can be mentioned.

The amino acid sequences represented by SEQ ID NO: 60 (PRT15), SEQ ID NO: 61 (PRT16), SEQ ID NO: 62 (PRT17), SEQ ID NO: 63 (PRT18), SEQ ID NO: 64 (PRT19), and SEQ ID NO: 65 (PRT20) are , SEQ ID NO: 46 (PRT1), SEQ ID NO: 47 (PRT2), SEQ ID NO: 48 (PRT3), SEQ ID NO: 49 (PRT4), SEQ ID NO: 50 (PRT5), and SEQ ID NO: 51 (PRT6), respectively. A tag sequence containing the amino acid sequence shown in SEQ ID NO: 70 is introduced at the N-terminus of . In addition, the amino acid sequences shown in SEQ ID NO: 66 (PRT21), SEQ ID NO: 67 (PRT22), SEQ ID NO: 68 (PRT23), and SEQ ID NO: 69 (PRT24) are SEQ ID NO: 52 (PRT7) and SEQ ID NO: 53 ( PRT8), SEQ ID NO: 54 (PRT9), and SEQ ID NO: 55 (PRT10) with a tag sequence including the amino acid sequence of SEQ ID NO: 70 introduced at the N-terminus.

The artificial fibroin of (iii) may have only the amino acid sequence shown in SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65.

SEQ ID NO: 60 (PRT15), SEQ ID NO: 61 (PRT16), SEQ ID NO: 62 (PRT17), SEQ ID NO: 63 (PRT18), SEQ ID NO: 64 (PRT19), or SEQ ID NO: 65 (PRT20) The GPGXX motif content of the artificial fibroin is 40.2%, 39.9%, 39.9%, 39.7%, 39.3%, and 38.6%, respectively, all of which are 10% or more. .

(iv) The artificial fibroin may have only the amino acid sequence shown in SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69.

The GPGXX motif content of the artificial fibroin containing the amino acid sequence shown in SEQ ID NO: 66 (PRT21), SEQ ID NO: 67 (PRT22), SEQ ID NO: 68 (PRT23), or SEQ ID NO: 69 (PRT24) is 39.9%, respectively. , 39.9%, 39.3%, and 38.6%, all of which are 10% or more.

The artificial fibroin described above may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

[Nucleic acid]
A nucleic acid according to one embodiment encodes the artificial fibroin. Specific examples of nucleic acids are amino acids represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55 Encodes artificial fibroin containing sequences, or artificial fibroin or the like in which the amino acid sequence (tag sequence) represented by SEQ ID NO: 70 or SEQ ID NO: 71 is bound to either or both of the N-terminus and C-terminus of these amino acid sequences Nucleic acids are included.

A nucleic acid according to one embodiment hybridizes under stringent conditions to a complementary strand of the artificial fibroin-encoding nucleic acid, and has formula 1: [(A) _n motif-REP] _m or formula 2: [( A) _n -motif-REP] _m -(A) A nucleic acid encoding an artificial fibroin comprising a domain sequence represented by _{the n-} motif. The above domain sequence of the artificial fibroin encoded by the nucleic acid has an amino acid sequence corresponding to the insertion of a cysteine residue into REP, compared to naturally-occurring fibroin.

"Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. "Stringent conditions" may be any of low stringent conditions, medium stringent conditions and high stringent conditions. Low stringency conditions means that hybridization occurs only when there is at least 85% or more identity between the sequences. Conditions for hybridization include: Moderately stringent conditions means that hybridization occurs only when there is at least 90% identity between the sequences, for example, using 5xSSC containing 0.5% SDS at 50°C. Conditions for hybridization include: High stringency conditions means that hybridization occurs only when there is at least 95% or more identity between the sequences. Conditions for hybridization include:

[Host and expression vector]
An expression vector according to one embodiment comprises the nucleic acid sequence described above and one or more regulatory sequences operably linked to the nucleic acid sequence. A regulatory sequence is a sequence that controls expression of a recombinant protein in a host (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected according to the type of host. The type of expression vector can be appropriately selected from plasmid vectors, virus vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, etc., depending on the type of host.

A host according to one embodiment is transformed with the above expression vector. As hosts, both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

As the expression vector, those capable of autonomous replication in host cells or capable of integration into the chromosome of the host and containing a promoter at a position at which the nucleic acid according to one embodiment can be transcribed are preferably used.

When prokaryotes such as bacteria are used as hosts, the expression vector according to one embodiment is capable of autonomous replication in the prokaryote, and at the same time includes a promoter, a ribosome binding sequence, a nucleic acid according to one embodiment, and a transcription termination sequence. It is preferably a vector containing A gene controlling a promoter may be included.

Examples of prokaryotes include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas.

Examples of microorganisms belonging to the genus Escherichia include Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BLR (DE3) (Merck Millipore), Escherichia coli DH1, Escherichia · Coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC 23506), Escherichia coli KY3276, Escherichia coli MC1000, Escherichia coli MG1655 (ATCC 47076), Escherichia coli No. 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 ( ATCC 27325), Escherichia coli XL1-Blue, and Escherichia coli XL2-Blue.

Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus borsterensis, Brevibacillus centropolus Brevibacillus formosus, Brevibacillus imvocatus, Brevibacillus latyrosporus, Brevibacillus limnophilus, Brevibacillus parabrevis , Brevibacillus reuszeri, Brevibacillus thermoluvar, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevibacillus brevis Bacillus brevis 47-5Q (JCM8975), Brevibacillus choshinensis HPD31 (FERM BP-1087), Brevibacillus choshinensis HPD31-S (FERM BP-6623), Brevibacillus choshinensis HPD31-OK (FERM BP- 4573), and Brevibacillus choshinensis SP3 strain (manufactured by Takara).

Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens ATCC14460, Serratia entomophila, Serratia ficaria, Serratia fonticola, and Serratia grimesi. (Serratia grimesii), Serratia proteamaculans, Serratia odorifera, Serratia polymuthica, and Serratia rubidae.

Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens.

Examples of microorganisms belonging to the genus Microbacterium include Microbacterium Ammoniaphyllum ATCC15354.

Examples of microorganisms belonging to the genus Brevibacterium include, for example, Brevibacterium divericatum (Corynebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, Brevibacterium inmariophyllum （Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ　ｉｍｍａｒｉｏｐｈｉｌｕｍ）ＡＴＣＣ１４０６８、ブレビバクテリウム・ラクトフェルメンタム（コリネバクテリウム・グルタミカムＡＴＣＣ１３８６９）ＡＴＣＣ１３６６５，ＡＴＣＣ１３８６９、ブレビバクテリウム・ロゼウムＡＴＣＣ１３８２５、ブレビバクテリウム・サッカロリティカム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ　ｓａｃｃｈａｒｏｌｙｔｉｃｕｍ）ＡＴＣＣ１４０６６、ブレビバクテリウム・thiogenitalis ATCC19240, Brevibacterium album ATCC15111, and Brevibacterium serinum ATCC15112.

Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6871 and ATCC6872, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, and Corynebacterium glutamicum ATCC14067.ラム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ａｃｅｔｏａｃｉｄｏｐｈｉｌｕｍ）ＡＴＣＣ１３８７０、コリネバクテリウム・アセトグルタミカムＡＴＣＣ１５８０６、コリネバクテリウム・アルカノリティカムＡＴＣＣ２１５１１、コリネバクテリウム・カルナエＡＴＣＣ１５９９１、コリネバクテリウム・グルタミカムＡＴＣＣ１３０２０，ＡＴＣＣ１３０３２，ＡＴＣＣ１３０６０、コリネバクテリウム・リリウムＡＴＣＣ１５９９０、 Corynebacterium melasecola ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), Corynebacterium herculis ATCC13868, and the like.

Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas brassicacerum, Pseudomonas fulva, and Pseudomonas Pseudomonas. (Pseudomonas sp.) D-0110 and the like.

Any method for introducing DNA into the host cell can be used as the method for introducing the expression vector into the host cell. For example, a method using calcium ions [Proc.　Natl.　Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP-A-63-248394), or the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979). can be mentioned.

Transformation of microorganisms belonging to the genus Brevibacillus, for example, the method of Takahashi et al. (J. Bacteriol., 1983, 156: 1130-1134) and the method of Takagi et al. -3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61: 202-203).

Examples of vectors for introducing a nucleic acid according to one embodiment (hereinafter simply referred to as "vectors") include pBTrp2, pBTac1, and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), and pSE280. (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese Patent Application Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [Escherichia coli JM109/pTrS32 (FERM08BP-54BP-54BP-5407)] )], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), JP-A-60-221091 Publication], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. , 172, 2392 (1990)], pGEX (manufactured by Pharmacia), and pET system (manufactured by Novagen).

When Escherichia coli is used as the host, suitable vectors include, for example, pUC18, pBluescriptII, pSupex, pET22b, and pCold.

Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP-A-2-31682), pNY700 (JP-A-4-278091), pHY4831 (J Bacteriol., 1987, 1239-1245), pNU200 (Shigezo Udaka, Journal of Japan Agricultural Chemistry 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU211R2L5 (JP-A-7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58: 525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177:745-749), pHT210 (JP-A-6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42:358-363), or Escherichia coli and microorganisms belonging to the genus Brevibacillus and pNCO2 (Japanese Unexamined Patent Application Publication No. 2002-238569), which is a shuttle vector for .

The promoter is not limited as long as it functions in the host cell. Examples thereof include promoters derived from Escherichia coli, phage, etc., such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. In addition, artificially designed and modified promoters such as a promoter in which two Ptrps are arranged in series (Ptrp×2), tac promoter, lacT7 promoter, let I promoter, and the like can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases). In the expression vector, a transcription termination sequence is not necessarily required for expression of the above nucleic acid, but it is preferred to place a transcription termination sequence directly under the structural gene.

Examples of eukaryotic hosts include yeast, filamentous fungi (molds, etc.), and insect cells.

As yeast, for example, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Schwanniomyces, the genus Pichia, the genus Candida , yeast belonging to the genus Yarrowia and Hansenula.より具体的には、サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ）、シゾサッカロマイセス・ポンベ（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）、クリベロマイセス・ラクチス（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ　ｌａｃｔｉｓ）、クリベロマイセス・マルキシアヌス（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ　ｍａｒｘｉａｎｕｓ）、トリコスポロン・プルランス（Ｔｒｉｃｈｏｓｐｏｒｏｎ　ｐｕｌｌｕｌａｎｓ）、シワニオマイセス・Schwanniomyces alluvius, Schwanniomyces occidentalis, Candida utilis, Pichia pastoris, Pichia angusta, Pichia morpha Pichia Pichiola (Pichia polymorpha), Pichia stipitis, Yarrowia lipolytica, and Hansenula polymorpha.

When yeast is used as a host cell, the expression vector usually contains an origin of replication (if amplification in the host is required) and a selectable marker for propagation of the vector in E. coli, a promoter for recombinant protein expression in yeast, and a It is preferred to include a terminator as well as a selectable marker for yeast.

When the expression vector is a non-integrating vector, it preferably further contains an autonomously replicating sequence (ARS). This can improve the stability of the expression vector in cells (Myers, AM, et al. (1986) Gene 45:299-310).

Examples of vectors when yeast is used as a host include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, and pPICZ B etc. can be mentioned.

The promoter is not limited as long as it can be expressed in yeast. Examples of promoters include promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter. , pGAP promoter, pGCW14 promoter, AOX1 promoter, and MOX promoter.

Any method for introducing DNA into yeast can be used as a method for introducing an expression vector into yeast. Examples of methods for introducing expression vectors into yeast include electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 ( 1984)), the lithium acetate method (J. Bacteriol., 153, 163 (1983)), and Proc. Natl. Acad. Sci. USA, 75, 1929 (1978), and the like.

Examples of filamentous fungi include Acremonium genus, Aspergillus genus, Ustilago genus, Trichoderma genus, Neurospora genus, Fusarium genus, Humicola genus, Penicillium genus, Myceliophtora genus, Botryts genus, Magnaporthe genus, Mucor genus, Metalhizium genus, Monascus genus, Rhizopus genus , and bacteria belonging to the genus Rhizomucoa.

Specific examples of filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus awamori Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）、アスペルギルス・サケ（Ａｓｐｅｒｇｉｌｌｕｓ　ｓａｋｅ）、アスペルギルス・ゾジエ（ソーヤ）（Ａｓｐｅｒｇｉｌｌｕｓ　ｓｏｊａｅ）、アスペルギルス・テュビゲンシス（Ａｓｐｅｒｇｉｌｌｕｓ　ｔｕｂｉｇｅｎｓｉｓ）、アスペルギルス・ニガー（Ａｓｐｅｒｇｉｌｌｕｓ　ｎｉｇｅｒ）、アスペルギルス・ニデュランス（Ａｓｐｅｒｇｉｌｌｕｓ　ｎｉｄｕｌａｎｓ）、アスペルギルス・パラシチクス（Ａｓｐｅｒｇｉｌｌｕｓ　ｐａｒａｓｉｔｉｃｕｓ）、アスペルギルス・フィクム（フィキュウム）（Ａｓｐｅｒｇｉｌｌｕｓ　ｆｉｃｕｕｍ）、アスペルギルス・フェニクス（Ａｓｐｅｒｇｉｌｌｕｓ　ｐｈｏｅｉｃｕｓ）、アスペルギルス・フォエチズス（フェチダス）（Ａｓｐｅｒｇｉｌｌｕｓ　ｆｏｅｔｉｄｕｓ）、アスペルギルス・フラーブス（Ａｓｐｅｒｇｉｌｌｕｓ　ｆｌａｖｕｓ）、アスペルギルス・フミガタス（Ａｓｐｅｒｇｉｌｌｕｓ　ｆｕｍｉｇａｔｕｓ）、アスペルギルス・ヤポニクス（ジャポニカス）（Ａｓｐｅｒｇｉｌｌｕｓ　ｊａｐｏｎｉｃｕｓ）、トリコデルマ・ビリデ（Ｔｒｉｃｈｏｄｅｒｍａ　ｖｉｒｉｄｅ）、トリコデルマ・ハージアヌム（Ｔｒｉｃｈｏｄｅｒｍａ　ｈａｒｚｉａｎｕｍ）、トリコデルマ・リーゼイ（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｓｅｅｉ）、クリソスポリウム・ルクノエンス（Ｃｈｒｙｓｏｓｐｏｒｉｕｍ　ｌｕｃｋｎｏｗｅｎｓｅ）、サーモアスクス（ Thermoascus, Sporotrichum, Sporotrichum cellulophilum, Talaromyces, Thielavia terrestris, Thielavia, Neurospora oxyspora, Neurospora oxy.ポーラス（Ｆｕｓａｒｉｕｍ　ｏｘｙｓｐｏｒｕｓ）、フザリウム・グラミネルム（Ｆｕｓａｒｉｕｍ　ｇｒａｍｉｎｅａｒｕｍ）、フザリウム・ベネナツム（Ｆｕｓａｒｉｕｍ　ｖｅｎｅｎａｔｕｍ）、フミコーラ・インソレンス（Ｈｕｍｉｃｏｌａ　ｉｎｓｏｌｅｎｓ）、ペニシリウム・クリゾゲナム（Ｐｅｎｉｃｉｌｌｉｕｍ　ｃｈｒｙｓｏｇｅｎｕｍ）、ペニシリウム・カマンベルティ（Ｐｅｎｉｃｉｌｌｉｕｍ　ｃａｍｅｍｂｅｒｔｉ）、ペニシリウム・カネセンス（Ｐｅｎｉｃｉｌｌｉｕｍ　ｃａｎｅｓｃｅｎｓ）、ペニシリウム・エメルソニ（Ｐｅｎｉｃｉｌｌｉｕｍ　ｅｍｅｒｓｏｎｉｉ）、ペニシリウム・フニクロスム（Ｐｅｎｉｃｉｌｌｉｕｍ　ｆｕｎｉｃｕｌｏｓｕｍ）、ペニシリウム・グリゼオロゼウム（Ｐｅｎｉｃｉｌｌｉｕｍ　ｇｒｉｓｅｏｒｏｓｅｕｍ）、ペニシリウム・パープロゲナム（Ｐｅｎｉｃｉｌｌｉｕｍ　ｐｕｒｐｕｒｏｇｅｎｕｍ）、ペニシリウム・ロケフォルチ（Ｐｅｎｉｃｉｌｌｉｕｍ　ｒｏｑｕｅｆｏｒｔｉ）、マイセリオフトラ・サーモFilm (Myceliophtaora thermophilum), Mucor ambigus, Mucor circinelloides, Mucor fragilis, Mucor himalis, Mucor inaequisporus , Mucor oblongiellipticus, Mucor racemosus, Mucor recurvus, Mocor saturninus, Mocor subtilismus, Ogataea Examples include Ogataea polymorpha, Phanerochaete chrysosporium, Rhizomucor miehei, Rhizomucor pusillus, and Rhizopus arhiza.

When the host is a filamentous fungus, the promoter may be a gene related to glycolysis, a gene related to constitutive expression, an enzyme gene related to hydrolysis, or the like. Specific examples of promoters when the host is a filamentous fungus include amyB, glaA, agdA, glaB, TEF1, xynF1tannasegene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, and catB, and the like.

Introduction of expression vectors into filamentous fungi can be performed using conventionally known methods. Methods for introducing expression vectors into filamentous fungi include, for example, the method of Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)], protoplast method [Mol. Gen. Genet. , 168:111 (1979)], competent methods [J. Mol. Biol. , 56:209 (1971)], electroporation, and the like.

Insect cells include, for example, Lepidoptera insect cells. More specifically, insect cells include Spodoptera frugiperda-derived insect cells such as Sf9 and Sf21, and Trichoplusia ni-derived insect cells such as High 5. .

Examples of vectors when insect cells are used as hosts include Baculovirus Expression Vectors such as Autographa californica nuclear polyhedrosis virus, which is a virus that infects insects of the family Mothidae. , A Laboratory Manual, WH Freeman and Company, New York (1992)).

When using insect cells as hosts, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W. H. Polypeptides can be expressed by methods described in Freeman and Company, New York (1992), and Bio/Technology, 6, 47 (1988). That is, after co-introducing a recombinant gene transfer vector and a baculovirus into insect cells to obtain a recombinant virus (expression vector) in the insect cell culture supernatant, the insect cells are further infected with the recombinant virus to produce a polypeptide. can be expressed. Examples of gene transfer vectors used in this method include pVL1392, pVL1393, and pBlueBacIII (both manufactured by Invitrogen).

Methods for co-introducing a recombinant gene transfer vector and baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-227075) and the lipofection method (Proc. Natl. Acad.Sci.USA, 84, 7413 (1987)).

A recombinant vector according to one embodiment preferably further contains a selection marker gene for transformant selection. For example, in E. coli, resistance genes to various drugs such as tetracycline, ampicillin, and kanamycin can be used as selectable marker genes. Recessive selectable markers that can complement genetic mutations involved in auxotrophy can also be used. In yeast, genes resistant to geneticin can be used as selectable marker genes, and selectable markers such as LEU2, URA3, TRP1, and HIS3, genes that complement gene mutations involved in auxotrophy can also be used. In filamentous fungi, selectable marker genes include niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6, 386-389, (1984)), sC (Gene, 84, 329-334, (1989)), ptrA (BiosciBiotechnol Biochem, 64, 1416-1421, (2000)), pyrG (Biochem Biophys Res Commun, 112, 284-289, (1983)), amdS (Gene, 26, 205-221, (1983)), aureobasidin resistance gene (Mol Gen Genet, 261, 290-296, (1999)), benomyl resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873, (1986)) and hygromycin-resistant genes (Gene, 57, 21-26, (1987)), marker genes selected from the group, leucine-requiring complementary genes, and the like. Moreover, when the host is an auxotrophic mutant strain, a wild-type gene that complements the auxotrophy can be used as the selectable marker gene.

A host transformed with an expression vector according to one embodiment can be selected by plaque hybridization, colony hybridization, or the like using a probe that selectively binds to the nucleic acid. As the probe, a partial DNA fragment amplified by PCR based on the sequence information of the above nucleic acid and modified with a radioisotope or digoxigenin can be used.

[Method for producing artificial fibroin]
Artificial fibroin can be produced by a method comprising the step of expressing the above nucleic acid in a host transformed with the above expression vector. As the expression method, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning, Second Edition. When expressed in yeast, animal cells, or insect cells, artificial fibroin can be obtained as a polypeptide to which a sugar or sugar chain has been added.

Artificial fibroin can be produced, for example, by culturing a host transformed with the expression vector in a culture medium, producing and accumulating the artificial fibroin according to the present embodiment in the culture medium, and collecting the artificial fibroin from the culture medium. can be done. The method of culturing the host in the culture medium can be carried out according to a method commonly used for culturing the host.

When the host is a prokaryote such as E. coli or a eukaryote such as yeast, the culture medium for the host contains a carbon source, a nitrogen source, an inorganic salt, etc. that can be assimilated by the host, Either a natural medium or a synthetic medium may be used as long as the medium can efficiently perform the above.

Any carbon source can be used as long as it can be assimilated by the host. Carbon sources include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. can be done.

Nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermented cells and their digests can be used.

As inorganic salts, for example, potassium dibasic phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

Prokaryotes such as E. coli or eukaryotes such as yeast can be cultured under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40°C. Culture time is usually 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using a lac promoter, and indole acrylic when culturing a microorganism transformed with an expression vector using a trp promoter. Acids and the like may be added to the medium.

Culture media for insect cells include, for example, commonly used TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by Life Technologies), ExCell400, ExCell405 (both manufactured by JRH Biosciences), and Grace's Insect Medium (Nature, 195, 788 (1962)).

Insect cells can be cultured for 1 to 5 days under conditions such as a culture medium with a pH of 6 to 7 and a culture temperature of 25 to 30°C. In addition, antibiotics such as gentamicin may be added to the culture medium as necessary during the culture.

When the host is a plant cell, the transformed plant cell can be cultured as it is, or it can be differentiated into plant organs and cultured. Examples of the medium for culturing the plant cells include commonly used Murashige and Skoog (MS) medium, White medium, or these medium supplemented with plant hormones such as auxin and cytokinin. can be used.

Animal cells can be cultured for 3 to 60 days under conditions such as a culture medium with a pH of 5 to 9 and a culture temperature of 20 to 40°C. Moreover, antibiotics such as kanamycin and hygromycin may be added to the medium as necessary during the culture.

Methods of producing artificial fibroin using a host transformed with the expression vector include a method of producing the artificial fibroin in the host cell, a method of secreting the artificial fibroin outside the host cell, and a method of producing it on the extracellular membrane of the host cell. There are methods, etc. Each of these methods can be selected by varying the host cell used and the structure of the artificial fibroin produced.

For example, when artificial fibroin is produced in the host cell or on the host cell extracellular membrane, the method of Paulson et al. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)), or by applying the methods described in JP-A-5-336963, WO 94/23021, etc. , can be modified to actively secrete artificial fibroin out of host cells. That is, artificial fibroin can be actively secreted outside the host cell by expressing a polypeptide containing the active site of artificial fibroin with a signal peptide added thereto using a genetic recombination technique.

Artificial fibroin produced by a host transformed with the above expression vector can be isolated and purified by methods commonly used for protein isolation and purification. For example, when artificial fibroin is expressed in a dissolved state in cells, the host cells are recovered by centrifugation after the completion of culture, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, or a Manton. The host cells are disrupted with a Gaulin homogenizer, Dynomill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for protein isolation and purification, that is, a solvent extraction method, a salting-out method using ammonium sulfate, etc., a desalting method, an organic solvent Precipitation method, diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei) and other resins, anion exchange chromatography, S-Sepharose FF (Pharmacia) and other resins, etc. Ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing A purified sample can be obtained by using the methods such as the above alone or in combination.

As the above chromatography, column chromatography using Phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), and Sephadex G-150 (Pharmacia Biotech) is preferably used.

In addition, when the artificial fibroin forms an insoluble form in the cells and is expressed, the host cells are similarly collected, crushed, and centrifuged to collect the insoluble form of the artificial fibroin as a precipitate fraction. The collected insoluble artificial fibroin can be solubilized with a protein denaturant. After this operation, a purified preparation of artificial fibroin can be obtained by the same isolation and purification method as described above.

When artificial fibroin or a derivative of artificial fibroin to which a sugar chain is added is secreted extracellularly, the artificial fibroin or its derivative can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.

[Binder]
Any type of binder can be used as long as it is a high-molecular polymer such as protein, peptide, polysaccharide, oligosaccharide, sugar polymer, fatty acid, fatty acid polymer, or polyphenol. These binders may or may not have the property of adhering to raw material fibers. The "property of adhering" is the property of binding different types of substances when they are brought close to each other by interaction, and includes "wetting" and "aggregation". The interaction is also called van der Waals force, surface tension, interfacial tension, electrostatic interaction, and the like.

After adhering to the raw material fibers, the adhesive binder immobilizes the bundle of the raw material fibers by enzymatic reaction to crosslink the binder and the raw material fibers, or between the binders, forming large-diameter artificial polypeptide fibers. In addition, the non-adhesive binder is prepared by bundling the raw material fibers in a cylindrical container or the like, and then by enzymatic reaction to cross-link the binder and the raw material fibers, or between the binders to fix the bundle of the raw material fibers, resulting in a large diameter. Form artificial polypeptide fibers.

Adhesive binders include polypeptides, peptides, proteins, polysaccharides, phenolic compounds, preferably fibroin (including natural and artificial), gelatin, collagen, casein glue, starch, kraft lignin, viscous polysaccharides. is mentioned.

Examples of non-adhesive binders include polypeptides, peptides, phenolic compounds, and polysaccharides. The polypeptide or peptide is preferably a polypeptide or peptide containing either tyrosine, lysine or cysteine. Phenolic compounds are preferably polyphenols, and polysaccharides are preferably polysaccharides to which phenols are bound (eg, sugar beet pectin, etc.).

In the method for producing an artificial polypeptide fiber according to the present embodiment, the amount of the binder used may be 0.001 to 10000% by mass, based on the mass of the raw material fiber. It is preferably 100% by mass, or 3 to 30% by mass.

〔enzyme〕
The enzyme used in the present embodiment may be any enzyme that can recognize at least one of the raw material fiber and the binder as a substrate for the enzyme, and may be an oxidoreductase or a protein cross-linking enzyme. Cross-linking enzymes also include acyltransferases and oxidoreductases.

Examples of protein cross-linking enzymes include polyphenol oxidase (tyrosinase), peroxidase, multi-copper oxidase (laccase, bilirubin oxidase, ascorbic acid oxidase, ceruloplasmin), transglutaminase, lysyl oxidase, protein disulfide isomerase, protein disulfide reductase, sulfhydryl oxidase. , lipoxygenase.

The protein cross-linking enzyme is preferably an oxidoreductase, and specific examples include polyphenol oxidase (tyrosinase), peroxidase, multicopper oxidase (laccase, bilirubin oxidase, ascorbic acid oxidase, ceruloplasmin), lysyl oxidase, and sulfhydryl oxidase. be done. More preferred are laccase, bilirubin oxidase, peroxidase and tyrosinase, and still more preferred are laccase, bilirubin oxidase and peroxidase.

More preferred enzymes are, for example, laccase, bilirubin oxidase, and combined use of peroxidase and glucose oxidase.

The oxidoreductase is not particularly limited as long as it can generate a reactive functional group (eg, aldehyde) by oxidizing or reducing the chemical structure of at least one of the binder and raw fiber. Examples of oxidoreductases include the following.

(1) Enzymes with low substrate specificity and containing metal ions in the active center (for example, multi-copper oxidases such as laccase)
(2) an enzyme that oxidizes the ε-amino group of a lysine residue in a protein to produce an aldehyde group (eg, lysyl oxidase)
(3) Enzymes that oxidize sulfhydryl groups of cysteine residues in proteins (e.g., sulfhydryl oxidase)
(4) Enzymes that oxidize phenolic hydroxyl groups of tyrosine residues in proteins to yield o-quinones (eg, tyrosinase)
(5) Enzymes that require hydrogen peroxide as an oxygen donor in oxidation reactions (for example, peroxidase, which may be used in combination with glucose oxidase to generate hydrogen peroxide)

Here, multi-copper oxidase is a group of enzymes that have a copper atom in the active center, extract (oxidize) electrons from various substrates such as polyphenols, methoxyphenols, diamines, bilirubin, and ascorbic acid, and reduce molecular oxygen. is a generic term for Enzymes with 2 to 8 copper atoms have been known so far, but there are variations depending on the state of the enzyme sample at the time of analysis and the analysis method. Multicopper oxidases include, for example, laccase, bilirubin oxidase, ascorbate oxidase, ceruloplasmin and the like.

Preferred combinations of binder and enzyme are, for example, polypeptide and laccase, polyphenol and laccase, polypeptide and bilirubin oxidase, polypeptide and peroxidase, polypeptide and peroxidase and glucose oxidase, more preferably silk fibroin and laccase, silk fibroin and bilirubin oxidase, silk fibroin and peroxidase, silk fibroin and peroxidase and glucose oxidase, gelatin and laccase.

Conventionally, cross-linking and adhesion of binders (especially proteins) have been performed using benzoquinone, glutaraldehyde, and formaldehyde as oxidizing agents (see, for example, Non-Patent Document 1). However, although benzoquinone and aldehydes are known to be oxidizing agents, they have an odor, are highly irritating to the skin and mucous membranes, and pose a physical burden on workers and the environment. Become. In addition, when aldehydes are used, a longer reaction time is required, and the resulting large-diameter artificial polypeptide fibers are colored. However, according to the method according to the present embodiment, enzymatic reaction can be used to increase the diameter, so there is no need to use the above-described oxidizing agent, and the burden on workers and the environmental load can be reduced. Also, in some embodiments, the high substrate specificity of the enzyme may reduce side reactions and reduce contamination.

The method according to the present embodiment may further comprise a step of preparing raw material fibers (thin-diameter artificial polypeptide fibers). The raw fibers obtained by this step can be bundled and crosslinked by an enzymatic reaction. In addition, in this step, for example, by referring to the method described in Patent Document 1, a thin artificial polypeptide fiber obtained through a spinning process from an artificial polypeptide may be used as a raw material fiber.

In the method according to the present embodiment, the enzyme reacts with at least one of the raw material fiber and the binder to generate a reactive functional group, which is crosslinked by chemically bonding with the surrounding raw material fiber or binder. A schematic diagram of the resulting artificial polypeptide fiber is shown in FIG. As shown in FIG. 1, raw material fibers 1 are bonded together via a binder 2 to form a single thick fiber 10 as a whole.

[Method for producing raw material fiber (thin artificial polypeptide fiber)]
Raw fibers can be produced by a known spinning method. That is, for example, when producing a raw material fiber containing protein as a main component, first, a protein (for example, modified fibroin) produced according to the above-described method is added to dimethyl sulfoxide (DMSO), N,N-dimethylformamide ( DMF), formic acid, or hexafluoroisopropanol (HFIP), if necessary, together with an inorganic salt as a dissolution accelerator, are added and dissolved to prepare a dope solution. Then, using this dope solution, spinning is performed by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning to obtain the desired raw material fiber. Preferred spinning methods include wet spinning or dry-wet spinning.

FIG. 7 is an explanatory diagram schematically showing an example of a spinning device for producing raw fibers. A spinning device 10 shown in FIG. 7 is an example of a spinning device for dry-wet spinning, and includes an extrusion device 1 , an undrawn yarn producing device 2 , a wet-heat drawing device 3 , and a drying device 4 .

A spinning method using the spinning device 10 will be described. First, the dope liquid 6 stored in the storage tank 7 is pushed out from the mouthpiece 9 by the gear pump 8 . On a lab scale, the dope solution may be filled into a cylinder and pushed out through a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied through the air gap 19 into the coagulation liquid 11 of the coagulation liquid bath 20, the solvent is removed, the protein is coagulated, and a fibrous coagulum is formed. Next, the fibrous coagulum is fed into hot water 12 in a drawing bath (washing bath) tank 21 and drawn. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14 . After that, the drawn fibrous coagulate is supplied to the drying device 4 and dried in the yarn path 22 to obtain the raw material fibers 36 as the wound yarn 5 . 18a-18g are thread guides.

The coagulating liquid 11 may be any solvent that can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulation liquid 11 may contain water as appropriate. The temperature of the coagulation liquid 11 is preferably 0 to 30.degree. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the nozzle 9, the extrusion rate is preferably 0.2 to 6.0 ml/hour, preferably 1.4 to 4.0 ml/hour. Time is more preferred. The distance over which the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the thread guide 18a to the thread guide 18b) should be sufficient for efficient removal of the solvent, for example, 200 to 500 mm. is. The undrawn yarn take-up speed may be, for example, 1 to 20 m/min, preferably 1 to 3 m/min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11 . The coagulation liquid bath 20 may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage as required.

The drawing performed when obtaining the raw material fiber may be, for example, the pre-drawing performed in the coagulating liquid tank 20 and the wet-heat drawing performed in the drawing bath 21, as well as the dry-heat drawing.

Wet heat stretching can be performed in hot water, in a solution of hot water added with an organic solvent or the like, or in steam heating. The temperature may be, for example, 50 to 90°C, preferably 75 to 85°C. In wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, preferably 2 to 8 times.

Dry heat drawing can be performed using an electric tubular furnace, a dry heat plate, or the like. The temperature may be, for example, 140°C to 270°C, preferably 160°C to 230°C. In the dry heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, preferably 1 to 4 times.

The wet heat drawing and the dry heat drawing may be performed independently, or they may be performed in multiple stages or in combination. That is, the first step drawing is performed by wet heat drawing, the second step drawing is performed by dry heat drawing, or the first step drawing is performed by wet heat drawing, the second step drawing is performed by wet heat drawing, and the third step drawing is performed by dry heat drawing. For example, wet heat stretching and dry heat stretching can be combined as appropriate.

The lower limit of the final draw ratio is preferably more than 1 times, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times with respect to the undrawn yarn (or pre-drawn yarn). 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less. , 12 times or less, 11 times or less, and 10 times or less.

In addition, the raw material fiber that repeatedly stretches and contracts in the wet state and the dry state described above is manufactured by, for example, shrinking the fiber obtained by spinning (that is, the fiber before contacting with water after spinning) with water. method. The shrinking step may include, for example, a step of irreversibly shrinking the fibers produced by the above spinning method (fibers after spinning but before contact with water) by contacting them with water (contacting step). The shrinking step may comprise, after the contacting step, drying the fibers to further shrink them (drying step).

The fiber diameter (cross-sectional diameter) of the raw material fiber may be, for example, 5 to 100 μm, 10 to 90 μm, 20 to 80 μm, 30 to 70 μm, or 40 to 60 μm.

[Second embodiment]
A second embodiment of the invention is an artificial polypeptide fiber obtainable by the method according to the first embodiment. In the artificial polypeptide fiber according to this embodiment, as shown in FIG. 1, raw material fibers 1 are bonded together via a binder 2 to form one thick fiber 10 as a whole.

The fiber diameter (cross-sectional diameter) of the artificial polypeptide fiber (large-diameter artificial polypeptide fiber) according to the present embodiment is, for example, 0.5 to 5.0 mm, 1.0 to 4.5 mm, 1.5 to 4.0 mm. 0 mm, 2.0-3.5 mm, or 2.5-3.0 mm. The number of raw fibers constituting the artificial polypeptide fiber usually matches the number of raw fibers to be bundled, for example, 5 to 100, 10 to 90, 20 to 80, 30 to 70, or 40 to 60. It can be adjusted within the scope of the book.

The artificial polypeptide contained in the artificial polypeptide fiber according to the present embodiment preferably contains a structural protein, more preferably fibroin, and even more preferably spider silk fibroin.

[Third Embodiment]
A third embodiment of the present invention is an adhesive for bonding fibers containing an artificial polypeptide together, the adhesive comprising a binder and an enzyme that reacts with at least one of the artificial polypeptide and the binder. is.

For the "fiber containing the artificial polypeptide" that is the target of the adhesive according to this embodiment, the definition of the raw material fiber described in the first embodiment can be referred to. Also, the description of the first embodiment can be referred to for the binder and the enzyme.

The adhesive according to the present embodiment may be a combination of a first composition containing a binder and a second composition containing an enzyme, or a composition containing both a binder and an enzyme. good. The former form is preferred when the binder is a reaction substrate for an enzyme.

The composition constituting the adhesive according to the present embodiment contains solvents, excipients, buffers, stabilizers, preservatives, preservatives, physiological saline, etc. in addition to binders and/or enzymes. good too. Solvents include dimethylsulfoxide (DMSO), diethylene glycol monoether, ethanol, methanol, isopropanol. As excipients, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used. Phosphate, citrate, acetate and the like can be used as buffers. Propylene glycol, ascorbic acid and the like can be used as stabilizers. Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. Ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. can be used as antiseptics.

The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[Production of modified fibroin]
(1) Construction of Expression Vector A modified fibroin (PRT799) having the amino acid sequence shown in SEQ ID NO: 15 and a modified fibroin (PRT918) having the amino acid sequence shown in SEQ ID NO: 37 were designed.

A nucleic acid encoding a protein with the designed amino acid sequence was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. These two types of nucleic acids were each cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was treated with restriction enzymes NdeI and EcoRI, cut out, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Expression of Modified Fibroin Escherichia coli BLR (DE3) was transformed with each of the obtained expression vectors. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that the OD ₆₀₀ was 0.005. The temperature of the culture solution was kept at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 mL of production medium (Table 5) was added so that the OD ₆₀₀ was 0.05, and the transformed E. coli was inoculated. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and culture was carried out for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the desired modified fibroin. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of the desired modified fibroin size band depending on the addition of IPTG. bottom.

(3) Purification of Modified Fibrons Cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The precipitate after washing was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH 7.0) to a concentration of 100mg/mL, and was incubated at 60°C for 30 minutes. Stir with a stirrer for 1 minute to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed with a freeze dryer, and a freeze-dried powder was recovered.

[Preparation of Raw Fiber]
(1) Preparation of Spinning Solution (Dope Solution) DMSO in which lithium chloride was dissolved to a concentration of 4% by mass was used as a solvent. was added to the solvent so that After melting with an aluminum block heater at 90° C. for 1 hour, insoluble matter and bubbles were removed to obtain a spinning solution (dope solution).

(2) Spinning A spinning solution was filled in a reserve tank and discharged from a monohole nozzle with a diameter of 0.1 or 0.2 mm into a 100% by mass methanol coagulation bath using a gear pump. The discharge rate was adjusted to 0.01-0.08 mL/min. After solidification, it was washed and stretched in a 100 wt% methanol washing bath. After washing and stretching, it was dried using a dry heat plate, and the obtained raw yarn (modified fibroin fiber) was wound up. The fiber diameter of the original yarn (raw material fiber) was 40 μm. The spinning conditions are as follows.

Nozzle hole diameter: 0.3mm
Coagulation liquid: 18% sodium sulfate aqueous solution Temperature of coagulation liquid: 50°C
Water wash bath temperature: 40°C
Hot roller temperature: 100°C

[Preparation of binder solution]
A 0.02 M sodium carbonate aqueous solution (400 mL) was added to silkworm cocoons (1 g), and the mixture was heated at 80 to 90° C. for 30 minutes or longer. The warmed liquid was filtered through a paper filter, washed with MilliQ water, and air-dried to obtain silk fibers. After adding 9.3 M lithium bromide aqueous solution (3 mL) to the obtained silk fibers (0.76 g), the mixture was heated at 60° C. for 5 hours or longer to completely dissolve the silk fibers. Insoluble materials were removed by centrifugation, and dialysis was repeated to exchange buffer 50 mM Tris-HCl buffer, pH 7.0, 150 mM sodium chloride. Thereafter, the buffer was replaced with a 50 mM Tris-hydrochloride buffer, pH 7.0, 150 mM sodium chloride buffer to obtain a silk fibroin solution. The obtained silk fibroin solution was divided, and various enzymes were added as shown in Table 6 so as to have a predetermined concentration to obtain binder solutions E1 to E3. For comparison, solutions NE1 (no binder or enzyme), NE2 (no enzyme), NE3 (no binder) and NE4 (no binder) were prepared.

As enzymes, laccase (manufactured by Amano Enzyme Co., Ltd.), bilirubin oxidase (manufactured by Amano Enzyme Co., Ltd.), glucose oxidase (manufactured by Amano Enzyme Co., Ltd.), and peroxidase (manufactured by Amano Enzyme Co., Ltd.) were used.

[Preparation of large-diameter artificial polypeptide fiber]
A bundle of 20-25 raw fibers (PRT918) was passed through each of seven 1 mL pipettes and capped while taped at both ends to stretch the fibers in each pipette. The seven binder solutions and buffers (50 mM Tris-HCl buffer of pH 7.0 with 150 mM sodium chloride added) shown in Table 6 were added to the seven pipettes thus obtained, and the mixture was added to Table 7 at room temperature. Incubated for the indicated time. When the solution solidified (the solution became cloudy and stopped flowing out of the tube), or after 3 hours had passed, the artificial polypeptide fiber was removed from the pipette and dried. Note that when solutions NE1, NE2, NE3 or NE4 were used, the solutions did not solidify even after 3 hours of incubation.

After that, when the artificial polypeptide fibers taken out from the pipette were observed with a scanning electron microscope, it was found that when solutions E1, E2, or E3 were used, the raw material fibers were crosslinked to each other during incubation, resulting in large-diameter artificial polypeptide fibers. It was confirmed that peptide fibers were formed. The large-diameter artificial polypeptide fibers thus obtained were referred to as Example 1, Example 2, and Example 3, respectively. On the other hand, among the solutions NE1, NE2, NE3 and NE4, only when the solution NE2 was added, some adhesion between the raw fibers was observed, but no adhesion between the fibers was observed with the other solutions. Also, when solutions NE1, NE2, NE3 or NE4 were used, the solutions did not solidify even after 3 hours of incubation. Among these artificial polypeptide fibers, fibers obtained using solutions NE1 and NE2 were designated as Comparative Examples 1 and 2, respectively.

Fig. 8 shows the actual procedure. Photograph 1 is a photograph showing how raw fibers (PRT918) are bundled. Photo 2 shows a state in which the bundled raw material fibers are placed in a pipette and one opening is closed with a seal. Photograph 3 is a photograph showing a state in which the other opening is closed with a seal. Photograph 4 is a photograph showing the obtained artificial polypeptide fiber having a large diameter. All of the thick artificial polypeptide fibers had a thickness of 1.6 mm.

A raw material fiber (PRT918) bundled with 20 to 25 fibers was used as Comparative Example 3.

[Evaluation of physical properties of artificial polypeptide fiber]
(1) Tensile test The strength of the fibers of Examples 1 to 3 and Comparative Examples 1 and 2 was evaluated and compared using a tensile tester (device name: INSTRON3345, manufactured by INSTRON) according to the following physical property evaluation criteria. A comparison was made with the stress of Example 3.

<Physical property evaluation>
A: When the stress of Comparative Example 3 is 100, the stress is 150 or more B: The stress is greater than the stress of Comparative Example 3 by 0.1 gf/den or more C: The stress α and the stress of Comparative Example 3 The difference from β (that is, α-β) is more than -0.1 gf / den and less than 0.1 gf / den and is comparable D: The stress is 0.1 gf / den or more smaller than the stress of Comparative Example 3

(2) Washing test 5 fibers of Examples 1 to 3 and 5 fibers of Comparative Examples 1 to 3 (5 bundles, that is, 25 fibers when not bonded) were placed in a glass container, and a reducing agent (Tris ( 2-Carboxyethyl)phosphine hydrochloride) was added and immersed and sonicated for 15 minutes. After that, it was visually observed whether or not the adhesion of the artificial polypeptide fiber was loosened, and evaluated according to the following evaluation criteria.

<Washing test>
A: All fibers remain fused after the washing test. B: 4 bundles of fibers remain fused after the washing test (one bundle is unfused).
C: 2 to 3 bundles of fused fibers remained after the washing test (2 to 3 bundles of fused bonds were unbonded).
D: 0 to 1 bundle of fibers remains fused after washing test (4 to 5 bundles of fused are unbonded)

Table 8 shows the results. In the physical property test, Examples 1 to 3 broke at once as the tensile strain was increased, but Comparative Examples 1 to 3 broke stepwise several times.

[Preparation of binder solution]
Freeze-dried pigskin-derived gelatin type A (manufactured by MP Biomedicals, LLC) was added to water to a predetermined concentration and heated to 60°C. Gelatin was completely dissolved by heating. A 20 mM sodium acetate aqueous solution was added as a buffer to the resulting gelatin aqueous solution to adjust the pH to 5.0.

The obtained gelatin solution (0.5 mL) was placed in a glass test tube, and laccase (manufactured by Amano Enzyme Co., Ltd.) was added as shown in Table 9 to obtain a binder solution E4.

(3) Production of large-diameter artificial polypeptide fiber A bundle of 20 to 25 raw material fibers was passed through a 1 mL pipette and capped while fixing both ends with tape so as to stretch the fibers in the pipette. A binder solution and 20 mM sodium acetate buffer pH 5.0 were added to the resulting pipette and incubated at 50° C. for 3 hours. During incubation, the raw fibers were cross-linked to each other to form large-diameter artificial polypeptide fibers (Example 4). At the timing when the solution solidified (a state in which the solution became cloudy and stopped flowing out of the tube) or after a certain period of time, the artificial polypeptide fiber was taken out from the pipette and dried. All the obtained artificial polypeptide fibers had a thickness of 1.6 mm.

(4) Washing test The obtained artificial polypeptide fiber was immersed in an aqueous solution containing a reducing agent (tris(2-carboxyethyl)phosphine hydrochloride) and subjected to ultrasonic waves for 15 minutes. Only the fiber bundle taken out from the pipette to which the binder solution E4 was added maintained adhesion between the fibers.

[Preparation of binder solution]
The silk fibroin solutions obtained in the same manner as described above were divided, and various enzymes were added to give predetermined concentrations as shown in Table 10 to obtain binder solutions E5 to E8. For comparison, solutions NE6 (no enzyme) and NE7 (no enzyme) were prepared.

[Preparation of large-diameter artificial polypeptide fiber]
Large-diameter artificial polypeptide fibers were produced in the same manner as in Examples 1 to 3, except that the concentration of the binder and the reaction time were changed. Table 11 shows the binder solutions and reaction times used. Comparative Examples 4-5 were prepared in the same manner as Example 5.

[Evaluation of physical properties of artificial polypeptide fiber]
(1) Tensile test The strength of the fibers of Examples 5 to 8 and Comparative Examples 4 to 5 was evaluated using a tensile tester (device name: INSTRON3345, manufactured by INSTRON) according to the above physical property evaluation criteria. 3 stress.

(2) Washing test Five fibers of Examples 5 to 8 and five fibers of Comparative Examples 4 to 5 (5 bundles when not bonded, that is, 25 fibers) were placed in a glass container, and a reducing agent (Tris ( 2-Carboxyethyl)phosphine hydrochloride) was added and immersed and sonicated for 15 minutes. After that, it was visually observed whether or not the adhesion of the artificial polypeptide fiber was loosened, and evaluated according to the evaluation criteria described above.

Table 12 shows the results. Examples 5-8 had a stress greater than that of Comparative Example 3 by 0.1 gf/den or more, and all fibers remained fused after the washing test.

[Preparation of binder solution]
A binder solution E9 was obtained by adding a 50 mM Tris-HCl buffer, pH 7.0, 150 mM sodium chloride buffer to commercially available sugar beet-derived pectin as shown in Table 13 instead of silk fibroin.

[Preparation of large-diameter artificial polypeptide fiber]
Large-diameter artificial polypeptide fibers were produced in the same manner as in Example 1, except that the concentration of the binder and the reaction time were changed. Table 14 shows the binder solutions and reaction times used. Comparative Example 6 was prepared in the same manner as Example 8.

[Evaluation of physical properties of artificial polypeptide fiber]
(1) Washing test Five fibers of Example 9 and five fibers of Comparative Example 6 (5 bundles, ie, 25 fibers when not bonded) were placed in a glass container, and a reducing agent (tris(2-carboxyethyl ) phosphine hydrochloride) was added and immersed, and ultrasonic waves were applied for 15 minutes. After that, it was visually observed whether or not the adhesion of the artificial polypeptide fiber was loosened, and evaluated according to the evaluation criteria described above.

Table 15 shows the results. In Example 9, all fibers remained fused after the wash test, but in Comparative Example 6, no adhesive effect was observed.

[Preparation of binder solution]
Binder solutions E9 to E11 were obtained by adding a buffer of 50 mM Tris-HCl buffer, pH 7.0, 150 mM sodium chloride to chemical oxidants (glutaraldehyde, formaldehyde) as shown in Table 16 instead of the enzyme.

[Preparation of large-diameter artificial polypeptide fiber]
Large-diameter artificial polypeptide fibers were produced in the same manner as in Example 1, except that the concentration of the binder and the reaction time were changed. Table 17 shows the binder solutions and reaction times used.

[Evaluation of physical properties of artificial polypeptide fiber]
(1) Tensile test The strength of the fibers of Comparative Examples 7 to 9 was evaluated using a tensile tester (device name: INSTRON3345, manufactured by INSTRON) according to the physical property evaluation criteria described above, and compared with the stress of Comparative Example 3. .

(2) Washing test 5 fibers of Comparative Examples 7 to 9 (5 bundles when not bonded, ie 25 fibers) were placed in a glass container, and a reducing agent (tris(2-carboxyethyl)phosphine hydrochloride) was added. The containing aqueous solution was added and immersed, and ultrasonic waves were applied for 15 minutes. After that, it was visually observed whether or not the adhesion of the artificial polypeptide fiber was loosened, and evaluated according to the above evaluation criteria.

The results are shown in Table 18. When chemical oxidizing agents were used instead of enzymes, silk fibroin was solidified, but the reaction rate was slow and longer reaction times were required. In addition, the results of the washing test were inferior, and the fibers were colored.

Claims

A step of bundling a plurality of raw fibers containing an artificial polypeptide;
a step of contacting the obtained bundle of raw material fibers with a composition containing a binder and an enzyme;
A method of making an engineered polypeptide fiber, comprising:
The method for producing an artificial polypeptide fiber according to claim 1, wherein the enzyme includes an oxidase.
The method for producing an artificial polypeptide fiber according to claim 2, wherein the oxidase is at least one selected from the group consisting of laccase, bilirubin oxidase, glucose oxidase, and peroxidase.
The method for producing an artificial polypeptide fiber according to any one of claims 1 to 3, wherein the artificial polypeptide contains a structural protein.
The method for producing an artificial polypeptide fiber according to any one of claims 1 to 3, wherein the artificial polypeptide contains fibroin.
The method for producing an artificial polypeptide fiber according to any one of claims 1 to 3, wherein the artificial polypeptide contains spider silk fibroin.
An artificial polypeptide fiber produced by the method according to any one of claims 1 to 3.
An artificial polypeptide fiber produced by the method according to claim 4.
An artificial polypeptide fiber produced by the method according to claim 5.
An artificial polypeptide fiber produced by the method according to claim 6.
An artificial polypeptide fiber in which multiple raw fibers containing artificial polypeptides are joined together via a binder.
The artificial polypeptide fiber according to claim 11, wherein said artificial polypeptide comprises a structural protein.
The artificial polypeptide fiber according to claim 11, wherein said artificial polypeptide comprises fibroin.
The artificial polypeptide fiber according to claim 11, wherein the artificial polypeptide comprises spider silk fibroin.
An adhesive for adhering fibers comprising engineered polypeptides together, comprising:
An adhesive comprising a binder and an enzyme that reacts with at least one of the artificial polypeptide and the binder.
The adhesive according to claim 15, wherein the enzyme is an oxidase.
The adhesive according to claim 16, wherein the oxidase is at least one selected from the group consisting of laccase, bilirubin oxidase, glucose oxidase, and peroxidase.
The adhesive according to any one of claims 15 to 17, wherein said artificial polypeptide comprises a structural protein.
The adhesive according to any one of claims 15 to 17, wherein said artificial polypeptide comprises fibroin.
The adhesive according to any one of claims 15 to 17, wherein the artificial polypeptide comprises spider silk fibroin.