WO2020067554A1

WO2020067554A1 - Production method for molded body and structural protein molded body

Info

Publication number: WO2020067554A1
Application number: PCT/JP2019/038435
Authority: WO
Inventors: アリナクダシェワ; 本章渡邉; 有希中山; 伸治平井
Original assignee: Ｓｐｉｂｅｒ株式会社; 小島プレス工業株式会社; 国立大学法人室蘭工業大学
Priority date: 2018-09-28
Filing date: 2019-09-27
Publication date: 2020-04-02
Also published as: JPWO2020067554A1

Abstract

This production method for a molded body includes: a molding step for heating and applying pressure to a composition that includes a structural protein and has a water content of at least 6% to obtain a molded body; and a rolling step for rolling the molded body obtained in the molding step at a rolling reduction of at least 60%.

Description

Method for producing molded article and structural protein molded article

The present invention relates to a method for producing a molded article and a structural protein molded article.

試み Attempts have been made to replace metal materials with organic materials for the purpose of reducing weight, reducing costs, facilitating molding, and the like. For example, Patent Literature 1 describes a technique for obtaining a molded article by applying pressure to a composition containing a powdery spider silk protein. Patent Literature 2 describes a technique for obtaining a higher-strength molded article by adding water to a powdery spider silk protein, applying pressure, and then forming the molded article, followed by drying.

International Publication No. WO 2017/047504 International Publication No. WO2018 / 043698

成形 A molded article manufactured by the above-mentioned conventional technique may cause brittle fracture with relatively small strain. For example, any of those molded articles may cause brittle fracture at a strain of several percent or less. Therefore, there is a need for a structural protein molded article that is less likely to cause brittle fracture.

The present invention provides a method for producing a molded article and a structural protein molded article that can obtain a molded article that contains a structural protein and is less likely to cause brittle fracture.

The present invention provides the following [1] to [6].
[1] A composition containing a structural protein and having a water content of 6% or more is heated and pressurized to form a molded article, and a molding step is carried out. A rolling step of rolling at a rolling reduction of:
[2] The method for producing a molded article according to [1], further comprising a moisture content adjusting step of adjusting the moisture content of the composition containing the structural protein to 6% or more before the molding step.
[3] The method for producing a molded article according to [1] or [2], wherein the structural protein is spider silk fibroin.
[4] A structural protein molded article having a breaking strain due to bending of 5.3% or more.
[5] A structural protein molded article having a strain energy due to bending of 600 J / m ³ or more.
[6] The structural protein molded article according to [5], wherein the structural protein is spider silk fibroin.

The above molded product is biodegradable because it uses structural protein as a raw material. Further, in the method for producing a molded article, a composition having a moisture content of 6% or more is heated and pressed to obtain a molded article (molding step), and the molded article is further rolled with a rolling reduction of 60% or more (rolling). Step 2). Due to these characteristics, this manufacturing method makes it possible to obtain a molded product that is less likely to cause brittle fracture.

The present invention makes it possible to obtain a molded article containing a structural protein and hardly causing brittle fracture.

FIG. 1 is a schematic diagram showing an example of the domain sequence of a modified fibroin. FIG. 2 is a diagram showing the distribution of z / w (%) values of naturally occurring fibroin. FIG. 3 is a diagram showing the distribution of x / y (%) values of naturally occurring fibroin. FIG. 4 is a schematic diagram showing an example of the domain sequence of a modified fibroin. FIG. 5 is a schematic diagram showing an example of the domain sequence of a modified fibroin. FIG. 6 is a graph showing a test result of a bending test of the molded bodies according to Comparative Examples 1 to 4. FIG. 7 is a graph showing the test results of the bending test of the molded bodies according to Comparative Examples 5 to 7 and the examples.

Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments.

製造 The method for producing a molded article according to the present embodiment is a method for producing a molded article containing a structural protein. In the method for producing a molded article according to the present embodiment, a composition containing a structural protein is used.

[Structural protein]
A structural protein is a protein having a role of constructing a biological structure, and is different from functional proteins such as enzymes, hormones, and antibodies. Examples of structural proteins include naturally occurring structural proteins such as naturally occurring fibroin, collagen, resilin, elastin, and keratin. Fibroin produced by insects and spiders is known as a naturally occurring fibroin. In this embodiment, the structural protein preferably includes a spider silk protein.

構造 The structural protein according to the present embodiment is preferably spider silk fibroin. Spider silk fibroin includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. That is, spider silk fibroin includes modified spider silk fibroin.

(Modified fibroin)
The modified fibroin according to this embodiment has a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. Including proteins. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.

As used herein, the term “modified fibroin” means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be the same as the amino acid sequence of naturally occurring fibroin. The term “naturally-derived fibroin” as used herein is also represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. Is a protein containing the domain sequence to be determined.

"Modified fibroin" may be a directly used amino acid sequence of a naturally occurring fibroin, or a modified amino acid sequence based on the amino acid sequence of a naturally occurring fibroin (for example, cloned naturally occurring fibroin). The amino acid sequence may be modified by modifying the gene sequence of fibroin), or may be artificially designed and synthesized without using naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence may be used). Which have a desired amino acid sequence by chemical synthesis).

As used herein, the term “domain sequence” refers to a crystalline region unique to fibroin (typically, corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence). The amino acid 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 indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues. (A) The _number of amino acid residues in the _n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16 . (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, and is 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 of only alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. The 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.

The modified fibroin according to the present embodiment has, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned natural fibroin gene sequence. Can be obtained by performing the following modification. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods {in} Enzymology, 100, 448 (1983).

Naturally occurring fibroin is a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. Yes, specifically, for example, fibroin produced by insects or spiders.

Examples of the fibroin produced by insects include Bombyx @ mori, Bombyx @ mandarina, natural silkworm (Antheraea @ yamamai), tussah (Anterea @ pernii), and maple silkworm (Erioganyerii). ), Silkworms produced by silkworms (Samia cynthia), chestnut worms (Caligura japonica), tussah silkworms (Antheraea mylitta), silkworms such as moga silkworms (Antheraea assama), and silkworms produced by larvae of the hornet beetle Hornet silk protein.

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

Examples of the fibroin produced by spiders include spiders belonging to the genus Araneus (genus Araneus), such as Orion spider, Elder spider, Red-colored Spider, Blue-colored Spider, etc. Spiders belonging to the genus Argiope genus (Genus Pronus), such as spiders belonging to the genus Procarpus spp., Spiders belonging to the genus Pronus, and spider spiders belonging to the genus Cynotarachne, such as the genus Cyrtarachne, such as Torinofundamashi and Otorinofundamashi. Spiders belonging to the genus Ordgarius, such as spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Orbalis, and spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Ordgarius. Spiders belonging to the genus Argiope such as Argiope bruennichi, spiders belonging to the genus Argiope sp. Spiders belonging to the genus Spider (Genus Poltys) such as spiders belonging to the genus (Cytophora) and spiders belonging to the genus (Potys), spiders belonging to the genus Spiders (genus Cyclosa) such as the spiders belonging to the genus Cyclosa and spiders belonging to the genus Cygnus spp. Spider silk proteins produced by spiders belonging to the genus Chorizopes), and asina such as red-headed spiders, red-backed spiders, blue-backed spiders and urocore-red spiders Spiders belonging to the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucaegium, such as the spiders Argiope bruennichi and spiders spiders belonging to the genus Leucauge, such as the spiders belonging to the genus Nephila sp. Spiders belonging to the genus Dyschiriognatha, such as spiders belonging to the genus Menosira and spiders belonging to the genus Dyschiriognatha, such as the spiders belonging to the genus Latus and the spiders belonging to the genus Lastroconidae belonging to the genus Latus sp. Spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops (Euprosthenops) are produced. Spider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ｐｈ ), U37520 (base sequence)), major \ ampullate \ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number ABA45, amino acid sequence of A23) )), Major \ sample \ spidroin \ 1 [from Euprosthenops \ australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ97155 (base sequence)), and major \ sample \ spidroin \ 2 [Euprosus] ] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor \ silk \ protein \ 1 [Nephila \ clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor \ ampilatte [in] Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampoulate spidroin-like protein [Nephilengys Cruenata] (GenBank Accession No. ABR3727.1.

より More specific examples of naturally occurring fibroin include fibroin in which sequence information is registered in NCBI GenBank. For example, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" is described as a keyword in DEFINITION from among sequences including INV as DIVISION in the sequence information registered in NCBI @ GenBank. It can be confirmed by extracting a character string of a specific product from a sequence or CDS, and extracting a sequence in which a specific character string is described in TISSUE @ TYPE from SOURCE.

The modified fibroin according to this embodiment may be a modified silk (silk) fibroin (an amino acid sequence of a silk protein produced by a silkworm modified), and a modified spider silk fibroin (a spider silk protein produced by an arachnid). Modified amino acid sequence). As the modified fibroin, a modified spider silk fibroin is preferable.

Specific examples of the modified fibroin include a modified fibroin (first modified fibroin) derived from a large spinal cord marker protein produced in a spider's large ampullate gland, and a domain sequence having a reduced content of glycine residues. (A second modified fibroin), (A) a modified fibroin having a domain sequence with a reduced content of the _n motif (a third modified fibroin), a glycine residue content, and (A) _n Modified fibroin having a reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence containing a region having a large hydrophobicity index (fifth modified fibroin), and glutamine residue content Modified fibroin having a reduced domain sequence (sixth modified fibroin).

The first modified fibroin includes a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, the number of amino acid residues of 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 still more preferable, an integer of 4 to 16 is still more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the first modified 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 more preferably 20 to 100 residues. Is even more preferable, and even more preferably 20 to 75 residues. The first modified fibroin has the total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m The total number is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more.

The first modified fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m , and has a C-terminal sequence represented by any 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 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is The amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.

As more specific examples of the first modified fibroin, (1-i) an amino acid sequence represented by SEQ ID NO: 4 (recombinant \ spider \ silk \ protein \ ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroin comprising an amino acid sequence having at least% sequence identity. The sequence identity is preferably 95% or more.

The amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human \ rhinovirus @ 3C protease) recognition site at the N-terminus is added. The 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue. The amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.

The modified fibroin of (1-i) may have an amino acid sequence represented by SEQ ID NO: 4.

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

The second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine, as compared with a naturally-derived fibroin. At least one motif sequence selected from the group consisting of at least one glycine residue in one or more of the motif sequences has been replaced with another amino acid residue. You may.

は In the second modified fibroin, the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.

The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the (A) _n motif located at the most C-terminal side to the domain sequence 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 is represented by z, From, when the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is defined as w, z / w is 30% or more; It may have an amino acid sequence of 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 more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).

２ The second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further 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 XGGX below.

The method of calculating z / w will be described in more detail. First, in a fibroin containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m (modified fibroin or naturally occurring fibroin), (A) _n located closest to the 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-terminal 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 (there is no duplication), z is 50 × 3 = 150. Further, for example, when there is an X (center X) included in two XGXs as in the case of an amino acid sequence consisting of XGXGX, calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues are used. is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminus to the C-terminus of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (the (A) _n motif located at the most C-terminal side is excluded). Next, z / w (%) can be calculated by dividing z by w.

Here, z / w in naturally occurring fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Of all the extracted fibroins, a naturally-derived one containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m and containing 6% or less of a GGX amino acid sequence in the fibroin Z / w was calculated from the amino acid sequence of fibroin by the above-described calculation method. The result is shown in FIG. The horizontal axis in FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z / w of the naturally derived fibroin is less than 50.9% (the highest one is 50.86%).

In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, and 70% or more. Is still more preferred, and even more preferably 80% or more. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

The second modified fibroin is modified, for example, by replacing at least a part of the base sequence encoding a glycine residue from the cloned natural fibroin gene sequence to encode another amino acid residue. Obtainable. At this time, as the glycine residue to be modified, a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more. Alternatively, for example, the amino acid sequence can be obtained by designing an amino acid sequence satisfying 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 each case, in addition to the modification corresponding to the substitution of another amino acid residue for the glycine residue in the REP from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. The amino acid sequence corresponding to insertion, addition, and / or addition may be modified.

The other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and 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 modified 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) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, Modified fibroin can be mentioned.

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

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

The modified fibroin of (2-i) may have an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified 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.

The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.

２The second modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.

Examples of the tag sequence include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography). Can be used for Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including a His tag sequence and a hinge sequence).

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

Furthermore, an “epitope tag” utilizing an antigen-antibody reaction can be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag. By using the epitope tag, the modified fibroin can be easily purified with high specificity.

Further, a tag sequence that can be cleaved by a specific protease can be used. By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified fibroin from which the tag sequence has been separated can also be recovered.

As more specific examples of the modified 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) Modified fibroin comprising a sequence or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .

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

The modified fibroin of (2-iii) may have an amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

The modified fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified 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.

The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.

{Circle around (2)} The second modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

The third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of the (A) _n motif as compared to a naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) _n motifs as compared to naturally occurring fibroin.

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

The third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal side toward the C-terminal one to three (A) _n motif every one (A) _n motif May have an amino acid sequence corresponding to the deletion of

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

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

The third modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side] -REP] The number of amino acid residues of REP in the unit is sequentially compared, and when the number of amino acid residues of REP having 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 When the maximum value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units that is 11.3 is x, and the total number of amino acid residues in the domain sequence is y In addition, 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 more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).

The method of calculating x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence is (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 an _n motif.

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

Next, for each pattern, the number of amino acid residues of each REP in two selected adjacent [(A) _n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of the other amino acid residues when the smaller number of the amino acid residues is set to 1. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is set to 1, the second REP is The ratio of the number of amino acid residues 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 having a smaller number of amino acid residues is set to 1, the fifth REP Is 30/20 = 1.5.

In FIG. 1, when the smaller number of amino acid residues is set to 1, the [(A) _n motif-REP] unit pair in which the ratio of the other amino acid residues is 1.8 to 11.3 is set. Shown by solid line. In the present specification, this ratio is called a jagged ratio. Assuming that the smaller number of amino acid residues is 1, the pair of [(A) _n motif-REP] units in which the ratio of the other amino acid residues is less than 1.8 or more than 11.3 is indicated by a broken line. Indicated.

In each pattern, the total number of amino acid residues of two adjacent [(A) _n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) _n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

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 modified fibroin, x / y is preferably at least 50%, more preferably at least 60%, further preferably at least 65%, and even more preferably at least 70%. Preferably, it is still more preferably at least 75%, particularly preferably at least 80%. The upper limit of x / y is not particularly limited, and may be, for example, 100% or less. When the indentation ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y. / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.

When the third modified fibroin is a modified fibroin in which at least seven of the (A) _n motifs present in a plurality in the domain sequence are composed of only alanine residues, x / y should be 46.4% or more. Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferred that there is. The upper limit of x / y is not particularly limited, and may be 100% or less.

Here, x / y in naturally occurring fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Among all the extracted fibroins, x / y was calculated from the amino acid sequence of the naturally occurring fibroin composed of the domain sequence represented by Formula 1: [(A) _n motif-REP] _m by the above calculation method. Was calculated. FIG. 3 shows the results when the indentation ratio is 1: 1.9 to 4.1.

横 The horizontal axis in FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is evident from FIG. 3, the x / y of the naturally derived fibroin is less than 64.2% (the highest is 64.14%).

The third modified fibroin, for example, deletes one or more of the sequence encoding the (A) _n motif from the cloned natural fibroin gene sequence such that x / y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed so that x / y is 64.2% or more based on the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. 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 further substituted, deleted, inserted and / or added. Amino acid sequence modification corresponding to the above may be performed.

As more specific examples of the third modified fibroin, (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-PRT525) -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, Modified fibroin can be mentioned.

The modified fibroin (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 17 differs from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin in that every two amino acids from the N-terminal side to the C-terminal side (A) _n The motif was deleted, and one [(A) _n motif-REP] was inserted before the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.

Ｘ The amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8-11.3. The value of x / y in the amino acid sequence represented by SEQ ID NO: 17 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%. The values of z / w 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. 1% and 70.0%.

The modified fibroin of (3-i) may be composed of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

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

The third modified fibroin may include the above-described tag sequence at one or both of the N-terminus and the C-terminus.

As more specific examples of the modified 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) Modified fibroin comprising a sequence or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding SEQ ID NO: 11 to the N-terminal 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. (Including a His tag sequence and a hinge sequence).

The modified fibroin of (3-iii) may have an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

The modified fibroin of (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified 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.

The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and is N-terminal to C-terminal. , The number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other The maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) _n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 is defined as x. Preferably, x / y is 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

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

The fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of a glycine residue in addition to the content of the (A) _n motif reduced as compared to a naturally-derived fibroin. Have The domain sequence of the fourth modified fibroin is at least one or more (A) _n motifs deleted, and further at least one or more glycine residues in the REP, as compared to naturally occurring fibroin. It can be said that it has an amino acid sequence equivalent to being replaced with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects and the like are as described for the second modified fibroin and the third modified fibroin.

As more specific examples of the fourth modified 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 represented by 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 And a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by. Specific embodiments of the modified fibroin comprising the amino acid sequence represented by 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 modified fibroin has a domain sequence in which one or more amino acid residues in REP have been replaced by amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and / or It may have an amino acid sequence locally including a region having a large hydrophobicity index, corresponding to insertion of one or more amino acid residues having a large hydrophobicity index therein.

領域 A region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

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

The fifth modified fibroin may have one or more amino acid residues in the REP replaced with amino acid residues having a higher hydrophobicity index, and / or one or more amino acids in the REP as compared to a naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, one or more amino acid residues may be substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be an amino acid sequence modification corresponding to the above.

The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned natural fibroin gene sequence, It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. In addition, for example, one or more hydrophilic amino acid residues in REP were replaced 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. Can be obtained by designing an amino acid sequence corresponding to the insertion of the amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more hydrophilic amino acid residues in REP were replaced 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. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) _n motif-REP] _m and extends from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence. In all REPs included in the sequence excluding the sequence from the domain sequence, the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p, When the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q, p / q is 6 .2% or more.

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

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by Formula 1: [(A) _n motif-REP] _m (Hereinafter, referred to as “sequence A”). First, in all REPs included in sequence A, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total 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 determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.

For example, when “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” are extracted at 20 locations (without duplication), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2 The region of .6 or more would contain 20 consecutive 4 amino acid residues (no duplication), and p would be 20 × 4 = 80. For example, when two “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of four consecutive amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “−1” is a deduction for the overlap). For example, in the case of the domain sequence shown in FIG. 4, there are seven consecutive “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without overlapping. 28. Also, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (the last (A) _n motif on the C-terminal side is not included). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

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

The fifth modified fibroin is, for example, one or a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the cloned natural fibroin is satisfied so as to satisfy the above-mentioned p / q conditions. Substituting a negative amino acid residue with a hydrophobic amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can also be obtained by designing an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP have been replaced by amino acid residues with a higher hydrophobicity index and / or one or more amino acid residues in the REP as compared to naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, a modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues may be performed. .

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.

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

The modified fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 19 consists of three amino acid residues every other REP, except for the domain sequence of the terminal at the C-terminal side, with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410). In this amino acid sequence, two amino acid sequences (VLI) were inserted, some glutamine (Q) residues were further substituted with serine (S) residues, and some C-terminal amino acids were deleted. The amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). is there. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8.

The modified fibroin of (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified 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 modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and is located at the most C-terminal side (A) _n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.

The fifth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus.

As more specific examples of the modified fibroin containing a tag sequence, (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) ) Modified 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 represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 correspond to the amino acid sequence represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).

The modified fibroin of (5-iii) may have an amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

The modified fibroin of (5-iv) 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 modified 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.

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located at the most C-terminal side (A) _n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.

(5) The fifth modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

The sixth modified fibroin has an amino acid sequence in which the content of glutamine residues is reduced as compared with naturally occurring fibroin.

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

場合 When the sixth modified fibroin contains a GPGXX motif in the REP, the content of the GPGXX motif is usually 1% or more, and 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.

In the present specification, the “GPGXX motif content” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif (modified fibroin or naturally occurring fibroin) Fibroin), the number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total number by 3 (ie, the total number of G and P in the GPGXX motif) is represented by s, and the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is represented (A) When the total number of amino acid residues of all REPs excluding the (A) _n motif is represented by t, the content of the GPGXX motif is calculated as s / t. It is.

In the calculation of the content ratio of the GPGXX motif, “the sequence obtained by removing the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) Sequence from _n motif to C-terminus of domain sequence (sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of fibroin, and m may be small. In this case (that is, when the domain sequence is short), the calculation result of the GPGXX motif content is affected, so that this effect is eliminated. When the “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “GPGXX motif”.

FIG. 5 is a schematic diagram showing the domain sequence of a modified fibroin. The method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 5 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are located at the most C-terminal side. (A) A sequence obtained by removing the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence ”(the sequence shown as“ region A ”in FIG. 5). 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 at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” (the sequence shown as “region A” in FIG. 5). ), The total number t of amino acid residues of all REPs excluding the (A) _n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, by dividing s by t, s / t (%) can be calculated. In the case of the modified fibroin of FIG. 5, 21/150 = 14.0%.

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

In the present specification, the “glutamine residue content” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the total number of glutamine residues contained in the region is defined as u, and the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence, and further (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 the motif. In the calculation of the glutamine residue content, the reason why the “sequence in which the sequence from the (A) _n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.

The sixth modified fibroin corresponds to the fact that its domain sequence has one or more glutamine residues in the REP deleted or replaced with other amino acid residues, as compared to the naturally occurring fibroin. It may have an amino acid sequence.

The “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can. Among them, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

The sixth modified fibroin preferably has a hydrophobicity of REP of -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, and 0.3 or more. Is still more preferable, and it is particularly preferable that it is 0.4 or more. The upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 0.7 or less.

In the present specification, “REP hydrophobicity” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is defined as v, and the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence. A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding _n motifs. In the calculation of the degree of hydrophobicity of the REP, the reason why the “sequence in which the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as follows. The same is true.

The sixth modified fibroin may have a domain sequence that is missing one or more glutamine residues in the REP and / or one or more glutamine residues in the REP, as compared to the naturally occurring fibroin. In addition to the modification corresponding to the substitution of with another amino acid residue, there may be an amino acid sequence modification equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues. .

The sixth modified fibroin may, for example, delete one or more glutamine residues in the REP from the cloned naturally occurring fibroin gene sequence and / or remove one or more glutamine residues in the REP. By substituting the amino acid residue with In addition, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As more specific examples of the sixth modified fibroin, (6-i) SEQ ID NO: 25 (Met-PRT988), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT889) -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 No. 42 (Met-PRT1028), or a modified fibroin comprising the amino acid sequence represented by (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 An amino acid represented by SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It can be mentioned modified fibroin comprising an amino acid sequence having a sequence at least 90% sequence identity.

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

アミノ酸 The amino acid sequence represented by SEQ ID NO: 30 is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence represented by SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 8 with VL and substituting the remaining Q with I.

The amino acid sequence represented by SEQ ID NO: 32 replaces all QQs in the sequence obtained by repeating twice the domain of the 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) with VF, In addition, the remaining Q is replaced with I.

アミノ酸 The amino acid sequence represented by SEQ ID NO: 41 (Met-PRT917) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with LI and replacing the remaining Q with V. The amino acid sequence represented by SEQ ID NO: 42 (Met-PRT1028) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with IF, and substituting the remaining Q with T.

The amino acid sequences represented by 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 all have glutamine residues. The group content is 9% or less (Table 2).

The modified fibroin of (6-i) has 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: It may consist of the amino acid sequence shown.

The modified fibroin of (6-ii) has 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: It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown. The modified 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 modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.

６The sixth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.

As more specific examples of the modified fibroin containing the tag sequence, (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 modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 44 (PRT1028), or ( 6-iv) The amino acid sequence represented by 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 % Modified amino acid sequence having an amino acid sequence having at least Mention may be made of the emissions.

The amino acid sequences represented by 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 correspond to SEQ ID NO: 25, respectively. SEQ ID NO: 11 at the N-terminal of the amino acid sequence represented by 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: Amino acid sequence (including a His tag sequence and a hinge sequence). Since only a tag sequence was added to the N-terminus, there was no change in the glutamine residue content, and 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 modified fibroin of (6-iii) has 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.

The modified fibroin of (6-iv) has 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 contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown. The modified 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 modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.

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

The modified fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. Modified fibroin having both of the following characteristics may be used.

The modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin. As used herein, the term "hydrophobic modified fibroin" refers to a value obtained by calculating the sum of the hydrophobicity indexes (HI) of all amino acid residues constituting the modified fibroin, and then dividing the total by the total number of amino acid residues. Modified fibroin having an (average HI) greater than 0. The hydrophobicity index is as shown in Table 1. Further, “hydrophilic modified fibroin” is a modified fibroin having an average HI of 0 or less. As the modified fibroin, a hydrophilic modified fibroin is preferred from the viewpoint of excellent combustion resistance, and a hydrophobic modified fibroin is preferred from the viewpoint of excellent moisture absorption and heat generation.

Examples of the hydrophobically modified fibroin include the amino acid sequence represented by 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: 33 or SEQ ID NO: 43, SEQ ID NO: 35, Modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44.

Examples of the hydrophilic modified fibroin include the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: Or the amino acid sequence represented by SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15 Modified fibroin comprising the amino acid sequence represented by the sequence, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

改変 The modified fibroin according to the present embodiment may contain only one kind of modified fibroin, or may contain two or more kinds of modified fibroins in combination.

(Structural proteins other than modified fibroin)
The structural protein may be a polypeptide derived from the above-mentioned natural type structural protein, that is, a recombinant polypeptide.

As a recombinant polypeptide of collagen, for example, a protein containing a domain sequence represented by Formula 3: [REP2] _o (where, in Formula 3, o represents an integer of 5 to 300. REP2 is Gly-X X and Y each represent an amino acid residue other than Gly. A plurality of REP2s may have the same amino acid sequence or different amino acid sequences.) Can be. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 45 can be mentioned. The amino acid sequence represented by SEQ ID NO: 45 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 obtained from the NCBI database (Genbank accession number of NCBI: CAA56335.1, GI: 3702452). The amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from residues 301 to 540.

As a recombinant polypeptide of resilin, for example, a protein containing a domain sequence represented by Formula 4: [REP3] _p (where _p represents an integer of 4 to 300 in Formula 4, and REP3 is Ser-J1 1 shows an amino acid sequence composed of J-Tyr-Gly-U-Pro, wherein J represents an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. Represents an arbitrary amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser .. A plurality of REP3s may have the same amino acid sequence or different amino acid sequences. Good)). Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 46 can be mentioned. In the amino acid sequence represented by SEQ ID NO: 46, in the amino acid sequence of resilin (NCBI Genbank, Accession No. NP 611157, Gl: 246654243), Thr at the 87th residue was replaced with Ser, and Asn at the 95th residue was replaced with Ser. Is a sequence obtained by adding the amino acid sequence (tag sequence) represented by SEQ ID NO: 49 to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced by Asp.

@Examples of elastin recombinant polypeptides include proteins having an amino acid sequence such as NCBI Genbank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, a protein comprising the amino acid sequence represented by SEQ ID NO: 47 can be mentioned. The amino acid sequence represented by SEQ ID NO: 47 corresponds to the amino acid sequence represented by SEQ ID NO: 11 (tag sequence) at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of Genbank accession number AAC98395 of NCBI. And a hinge sequence).

組換え Keratin recombinant polypeptides include, for example, Capra hircus type I keratin and the like. Specifically, a protein comprising the amino acid sequence represented by SEQ ID NO: 48 (the amino acid sequence of Genbank accession number ACY30466 of NCBI) can be mentioned.

(Method for producing modified fibroin)
Hereinafter, a method for producing a modified fibroin will be described as an example, but the following production method is similarly applicable to structural proteins other than modified fibroin. The modified fibroin according to any of the above embodiments may be, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.

方法 The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding a natural fibroin and modifying it by a genetic engineering technique, or a chemical synthesis method. can do. The method for chemically synthesizing nucleic acids is not particularly limited. For example, based on amino acid sequence information of fibroin obtained from the NCBI web database or the like, AKTA oligopilot plus１００10 / 100 (GE Healthcare Japan Co., Ltd.) Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like. At this time, to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence comprising an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is synthesized. May be.

The regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host. An inducible promoter that functions in a host cell and is capable of inducing the expression of a modified fibroin may be used as the promoter. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.

種類 The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. As the expression vector, those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed are suitably used.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.

好ましい Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like. Examples of microorganisms belonging to the genus Bacillus include, for example, Bacillus subtilis. Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

When a prokaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.

Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold). Examples of the yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

場合 When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. {Natl. {Acad. {Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, and the like.

As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .

The modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. The method of culturing the host in the culture medium can be performed according to a method usually used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.

The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolyzate, acetic acid and propionic acid Organic acids and alcohols such as ethanol and propanol can be used. As the nitrogen source, 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, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used. As the inorganic salts, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

培養 Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culturing time is usually 16 hours to 7 days. The pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

抗生 During the culture, if necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium. 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, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. An acid or the like may be added to the medium.

単離 The expressed and modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton. The host cells are crushed 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 isolating and purifying proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion-exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia). Electrophoretic methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Purification using methods such as alone or in combination It is possible to obtain the goods.

When the modified fibroin is expressed by forming an insoluble form in the cells, the host cells are similarly recovered, crushed, and centrifuged to collect the insoluble form of the modified fibroin as a precipitate fraction. The recovered insoluble form of the modified fibroin can be solubilized with a protein denaturant. After this operation, a purified sample of the modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.

[Method of manufacturing molded article]
The method for producing a molded article according to this embodiment includes, for example, a molding step of heating and pressurizing a composition containing a structural protein obtained as described above to obtain a molded article. In this molding step, the composition may have a moisture content of 6% or more, and preferably has a moisture content of 7.5% or more. The method for producing a molded article according to the present embodiment may include, before the molding step, a moisture percentage adjusting step of adjusting the moisture percentage of the composition to 6% or more, and preferably a moisture percentage adjusting to 7.5% or more. A rate adjusting step may be included. By performing the moisture content adjustment step first, it is possible to reliably obtain a molded article that is less likely to cause brittle fracture, regardless of the moisture content of the composition at the time when the structural protein is obtained. In the molded structural protein according to the present embodiment, the breaking strain due to bending is 5.3% or more. In the structural protein molded article according to the present embodiment, the stress due to bending can be 165 MPa or more (without causing brittle fracture). In the structural protein molded article according to the present embodiment, the strain energy due to bending is 600 J / m ³ or more.

The composition only needs to contain a structural protein. The composition may be only the structural protein, or may include the structural protein and optional additives (for example, a plasticizer, a colorant, a filler, a synthetic resin, and the like). It is preferable that the content of the additional component be 50% by mass or less of the total amount of the structural proteins. The composition is typically in the form of a powder (such as a lyophilized powder) or a fibrous (such as a fiber obtained by spinning). The molding composition may be a fusion of the composition comprising the structural protein in such a shape.

水分 The moisture content of the composition can be measured using a known moisture meter. Examples of the method for measuring the moisture content include various measurement methods such as a near infrared method, an electric resistance method, an electric capacity method, a dry weight method, and a chemical measurement method including the Karl Fischer method. The moisture content of the composition can be measured using any one of known measurement methods. For example, after the composition is sufficiently stirred and homogenized, a part of the composition may be taken out and the moisture content of the part may be measured. When adjusting the moisture content of the composition, the composition can be adjusted to a desired moisture content while the composition is placed in a predetermined atmosphere and dried or humidified.

(4) The moisture content of the composition at the start of the molding step may be 6% or more, preferably 7.5% or more. The moisture content of the composition at the start of the molding step is preferably less than 15%, more preferably less than 9%.

方法 The method for producing a molded article according to the present embodiment includes, for example, a molding step of heating and pressing the composition whose moisture content has been adjusted in the moisture content adjusting step to obtain a molded article. Note that the moisture adjustment step may be omitted. That is, in the molding step, a composition having a moisture content of 6% or more, preferably a composition having a moisture content of 7.5% or more, may be prepared, and this composition may be heated and pressed.

In the molding step, for example, a composition containing a structural protein is introduced into a mold, and the composition is molded while being heated and pressed to obtain a molded article (mold molded article). In the molding step, for example, a pressure molding machine can be used. Although the configuration of the pressure molding machine is not particularly limited, for example, a pressure molding machine having the same configuration as the pressure molding machine described in Patent Document 2 (WO 2018/043698) is used. obtain. The pressure molding machine has a function of heating a mold, for example. As for the method of heating and pressurizing, the method described in Patent Document 2 can be used. The pressure molding machine heats the mold inside by heating the mold. The pressure molding machine may be configured to measure, for example, the temperature of an internal compact. It should be noted that the present invention is not limited to the above-described apparatus and method, and other known molding methods and a method of heating and pressing a molded body may be employed.

加熱 The heating in the molding step is preferably performed, for example, at a mold temperature of 80 to 300 ° C, more preferably 100 to 180 ° C, and still more preferably 100 to 130 ° C. The pressurization is preferably performed at 5 kN or more, more preferably 10 kN or more, even more preferably 20 kN or more. After reaching the predetermined heating and pressurizing condition, the time for continuing the treatment under the condition (temperature keeping condition) is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, and further preferably 5 to 30 minutes. When the heat molding machine is configured to measure the temperature of the internal molded body, the heating in the molding step is preferably performed, for example, at a temperature of the molded body of 80 to 300 ° C, and 100 to 180 ° C. Is more preferable, and 100 to 130 ° C. is further preferable.

製造 The method for manufacturing a compact according to the present embodiment further includes a rolling step of rolling the compact obtained in the forming step. In this rolling step, the compact obtained in the compacting step is compressed and stretched. In the rolling step, for example, a stretching machine can be used. Although the configuration of the stretching machine is not particularly limited, for example, a stretching machine having two cylindrical rolls is used. In the rolling step, for example, the two rolls are respectively rotated and fed while applying pressure while sandwiching the formed body between the two rolls. Thereby, the whole of the molded body is compression-stretched. In this rolling step, the compact may or may not be heated. When heating a molded object while rolling, a heating stretching machine may be used.

では In the rolling step of the present embodiment, the compact may be rolled with a draft of 60% or more. In the rolling step of the present embodiment, preferably, the compact is rolled with a draft of 69.3% or more. In the present specification, the rolling reduction is a reduction rate of a plate thickness (a thickness of a formed body). In the rolling step, the higher the rolling reduction, the higher the strength of the formed body, but the rolling reduction can be determined according to the application. When a rolling mill having two rolls (upper roll and lower roll) is used, the rolling reduction of the compact can be set by determining the positions (intervals) of these rolls.

When heating is performed in the rolling step, the heating is preferably performed at 80 to 200 ° C, more preferably 100 to 180 ° C, and even more preferably 120 to 160 ° C.

The rolling step is not limited to the form using two rolls, but may be performed in another form capable of performing compression stretching. For example, the molding composition may be compression-stretched by placing the molding composition on the substrate using a stretching machine having a flat substrate and a roll, and rolling and pressing the roll from above. Alternatively, the molding composition may be compression-stretched by using a stretching machine having a flat upper mold and a lower mold, and applying pressure while sandwiching the molding composition between the upper mold and the lower mold. The setting of the rolling reduction is the same as in the case of two rolls. By determining the position (interval) between the upper member and the lower member, the rolling reduction of the molded body can be set.

成形 A molded article is manufactured through the moisture content adjusting step, the forming step, and the rolling step. By setting the moisture content of the composition and the rolling reduction of the molded body in the above-described ranges, a molded body that is unlikely to cause brittle fracture can be obtained. For example, a molded article that does not cause brittle fracture even with a large strain of several percent or more (for example, 5% or more) can be obtained. That is, a molded body that does not break or breaks even with a large strain of several percent or more (for example, 5% or more) can be obtained.

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

(1) Production of Strain Expressing Structural Protein Strain after obtaining the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P468804.1, GI: 11744415) derived from Nephila clavipes from GenBank web database, The amino acid sequence represented by SEQ ID NO: 13 was substituted with an amino acid residue, inserted and deleted for the purpose of improving the sex, and the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) was added to the N-terminus. A modified fibroin having a sequence (hereinafter, also referred to as “PRT410”) was designed.

Next, we commissioned synthesis of the gene encoding PRT410. As a result, a gene was obtained in which an NdeI site was added immediately upstream of the 5 'end of the gene and an EcoRI site was added immediately downstream of the 3' end. After cloning the gene into a cloning vector (pUC118), it was treated with NdeI and EcoRI with restriction enzymes, and recombined into the protein expression vector pET-22b (+).

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with the pET22b (+) expression vector containing the gene encoding PRT410 obtained above. Transformed E. coli, after 15 hours of culture in LB medium 2mL containing ampicillin, and the culture solution, the culture medium for the seed culture 100mL shown in Table 4 _was added so that _{OD 600} of 0.005. The temperature of the culture was maintained at 30 ° C., and culturing was continued for about 15 hours in a flask until the OD ₆₀₀ reached 5, to obtain a seed culture.

The obtained seed culture solution was added to a jar fermenter to which 500 mL of the production medium shown in Table 5 had been added so that the OD ₆₀₀ was 0.05. The temperature of the culture was maintained at 37 ° C., the pH was controlled to be constant at 6.9, and the culture was performed so that the dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration. Adekanol LG-295S (manufactured by ADEKA Corporation) was used as an antifoaming agent.

フィード Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, the culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, a 1M aqueous solution of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the target protein. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells. SDS-PAGE was performed using cells prepared from the culture solution before and after the addition of IPTG, and the expression of the target protein was confirmed by the appearance of a band of the target protein size dependent on the addition of IPTG.

(3) Purification of structural protein Two hours after the addition of IPTG, the recovered cells were washed with a 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM phenylmethylsulfonyl fluoride (PMSF), and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in an 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL. For 30 minutes with a stirrer 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 the dialysis was collected by centrifugation, and water was removed with a freeze dryer to collect a freeze-dried powder.

<Preparation of molded body>
Next, the water content of the obtained freeze-dried powder was adjusted. In order to produce the molded articles according to Comparative Examples 1 to 4, a heat-drying moisture meter (MS-70, manufactured by A & D Co., Ltd.) was used to reduce the weight change rate per minute to 0.01 wt% or less. The powder was dried at 130.degree. C. until the temperature reached 23.degree. C. and placed in an atmosphere of 23.degree. C. and 60% RH for 5 minutes to obtain a spider silk fibroin powder (structural protein powder) having a water content of 5.8%. Further, in order to produce the compacts according to Comparative Examples 5 to 7 and the examples, the powder was dried using the same apparatus and under the same conditions as above, and the powder was placed in an atmosphere of 23 ° C. and 60% RH for 7 minutes. A 7.5% spider silk fibroin powder (structural protein powder) was obtained.

Subsequently, the powder was heated and pressed using a press forming machine to obtain a formed body. The mold of this press molding machine is a mold having a rectangular through hole having a cross section of 35 mm × 15 mm. The powder was introduced into a through hole of a mold of a pressure molding machine, the mold was heated, and 40 kN was applied using a hand press (trade name: NT-100H-V09, manufactured by NPa System Co., Ltd.). (27.55 MPa). When the temperature of the molded body reached 130 ° C., the heating was stopped, and the molded body was cooled with a spot cooler (trade name: TS-25EP-1, manufactured by Trusco Nakayama Co., Ltd.). At this point, the compact was removed from the mold. In Comparative Examples 1 to 7 and Examples, the molding process is the same (common). For each of Comparative Examples 1 to 7 and Example, a rectangular molded article of 35 mm × 15 mm × 3 mm was obtained.

成形 Subsequently, the formed body was rolled using a stretching machine. In Comparative Examples 1 and 5, this rolling step was not performed, and the above-mentioned molded product was subjected to a bending test. Using a manual heating stretching machine (Model IMC-1989, manufactured by Imoto Seisakusho Co., Ltd.), each molded product according to Comparative Examples 2 to 4, 6 to 7 and Example was directly sandwiched between rolls and stretched. In the molded articles according to Comparative Examples 2 to 4 and 6 to 7, the rolling reduction (rate of reduction in plate thickness) was set to 25.4 to 58.8% (see Table 6 for details). In the molded article according to the example, the rolling reduction (rate of reduction in plate thickness) was 69.3%.

<Bending test>
A three-point bending test was performed on each of the molded bodies produced as described above using a table-top precision universal testing machine (Autograph AGS-X, manufactured by Shimadzu Corporation). The load cell used was 50 kN, the distance between fulcrums was 27 mm, and the measurement speed was 1 mm / min. Using the numerical data of the strain and the stress obtained by this test, the stress was integrated by the strain to calculate the strain energy.

結果 The results of the bending test are shown in Table 6, FIG. 6 and FIG. In Comparative Examples 1 to 6, fracture, ie, brittle fracture, occurred with a strain of less than 3%. In Comparative Example 7, fracture, that is, brittle fracture occurred at a strain of 5.2%. In the example, even when the strain was 10% (that is, even when the operation range of the autograph was exceeded), no fracture, that is, no brittle fracture occurred. That is, in the example, even if the distortion was 10%, the molded body was not broken. As in the example, a resin that was not easily broken when subjected to external force or impact was obtained. In Table 6, the stress at the time of plastic deformation is shown for the examples. The stress at a strain of 10% in the example was 219 MPa.

Claims

A molding step of heating and pressurizing the composition containing a structural protein and having a water content of 6% or more to obtain a molded article;
A rolling step of rolling the formed body obtained in the forming step at a rolling reduction of 60% or more.
2. The method for producing a molded article according to claim 1, further comprising a moisture content adjusting step of adjusting the moisture content of the composition containing the structural protein to 6% or more prior to the molding step.
方法 The method for producing a molded article according to claim 1 or 2, wherein the structural protein is spider silk fibroin.
構造 A structural protein molded article having a bending strain of 5.3% or more due to bending.
A structural protein molded article having a bending strain energy of 600 J / m 3 or more.
構造 The structural protein molded article according to claim 5, wherein the structural protein is spider silk fibroin.