JP7458619B2 - Fibroin fiber manufacturing method and fibroin solution - Google Patents

Description

The present invention relates to a method for producing fibroin fibers and a fibroin solution.

Fibroin fibers have attracted attention as a new material with high utility value in various industrial fields. Regenerated silk fibroin fibers and spider silk fibroin fibers are known examples of fibroin fibers. Many wet spinning and dry-wet spinning methods have been reported as methods for manufacturing these fibroin fibers. However, these manufacturing methods require the use of large amounts of liquid in the coagulation bath and water washing bath, and there are limitations to improving productivity.

Examples of the dry spinning method include, for example, a method in which fibers are formed by discharging a dope prepared by dissolving refined silkworm silk fibroin in a mixed organic solvent of calcium chloride and formic acid into the air from a syringe (Non-Patent Document 1); A method has been reported in which fibers are formed by discharging a dope in which refined silkworm silk fibroin is dissolved in hexafluoroisopropanol (HFIP) from a syringe into the air (Non-Patent Document 2).

Xiaoxiao Yue et al, MaterialsLetters, 128, 2014, pp.175-178 Andreas Koeppel et al, ACSBiomaterials Science & Engineering, 2017, 3, 226-237

However, it is difficult to obtain sufficient tensile strength with fibroin fibers obtained by conventional dry spinning methods.

Therefore, an object of the present invention is to obtain fibroin fibers having excellent tensile strength.

The present invention relates to the following inventions, for example.
(1) A dope solution containing fibroin and a solvent containing a first solvent as a main component and a second solvent having a boiling point higher than the boiling point of the first solvent as an additive component is spun by dry spinning. A method for producing fibroin fiber, comprising the step of:
(2) The method for producing fibroin fibers according to (1), wherein the mass ratio of the content of the first solvent to the content of the second solvent is 95/5 or more.
(3) The method for producing fibroin fibers according to (1) or (2), further comprising a step of stretching the fibers obtained in the above step in water.
(4) The fibroin fiber according to any one of (1) to (3), wherein the first solvent has a boiling point of 40°C or more and 120°C or less, and the second solvent has a boiling point of 80°C or more and 280°C or less. manufacturing method.
(5) The method for producing fibroin fibers according to any one of (1) to (4), wherein the fibroin is derived from insects or arachnids.
(6) The method for producing fibroin fibers according to any one of (1) to (5), wherein the fibroin is modified fibroin.
(7) A fibroin solution containing fibroin and a solvent containing a first solvent as a main component and a second solvent having a boiling point higher than the boiling point of the first solvent as an added component.
(8) The fibroin solution according to (7), wherein the mass ratio of the content of the first solvent to the content of the second solvent is 95/5 or more.
(9) The fibroin solution according to (7) or (8), wherein the first solvent has a boiling point of 40°C or more and 120°C or less, and the second solvent has a boiling point of 80°C or more and 280°C or less.
(10) The fibroin solution according to any one of (7) to (9), wherein the fibroin is derived from an insect or arachnid.
(11) The fibroin solution according to any one of (7) to (10), wherein the fibroin is modified fibroin.
(12) The fibroin solution according to any one of (7) to (11), which is used for dry spinning.

According to the present invention, it is possible to obtain fibroin fibers with excellent tensile strength.

FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 2 is a diagram showing the distribution of z/w (%) values of naturally-derived fibroin. FIG. 2 is a diagram showing the distribution of x/y (%) values of naturally-derived fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin.

Embodiments of the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[Fibroin solution]
The fibroin solution (also called a dope solution) of the present embodiment contains fibroin and a solvent. The solvent contains a first solvent as a main component and a second solvent as an additive component.

The first solvent is a solvent capable of dissolving fibroin, and preferably has a high solubility for fibroin. The first solvent is preferably a solvent capable of dissolving 6% or more by mass of fibroin at 25°C.

The boiling point BP1 of the first solvent may be, for example, 40°C or more, 45°C or more, 50°C or more, or 55°C or more, and may be 120°C or less, 110°C or less, 100°C or less, 90°C or less, 80°C or less, 70°C or less, or 60°C or less. In this specification, the boiling point means the boiling point under atmospheric pressure. Examples of the first solvent include hexafluoroisopropanol, perfluoro-t-butanol, trifluoroethanol, and trifluoroacetic acid.

The second solvent is a solvent having a boiling point BP2 higher than the boiling point BP1 of the first solvent. The solubility of the second solvent in fibroin is not particularly limited.

The difference (BP2-BP1) between the boiling point BP1 of the first solvent and the boiling point BP2 of the second solvent is, for example, 30°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, or 80°C or higher. , 90°C or higher, 100°C or higher, 110°C or higher, 120°C or higher, or 130°C or higher, for example, 200°C or lower.

The boiling point BP2 of the second solvent may be, for example, 80°C or higher, 100°C or higher, 120°C or higher, 140°C or higher, 160°C or higher, or 180°C or higher, and 280°C or lower, 260°C or lower, or 240°C. The temperature may be below 230°C, below 220°C, below 210°C, or below 200°C. Examples of the second solvent include N,N-dimethylformamide, formic acid, dimethylsulfoxide, N,N-dimethylacetamide, propylene carbonate, imidazole, 1-methylimidazole, ionic liquids (1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium diethyl phosphate, etc.).

The total content of the first solvent and the second solvent may be 50% by mass or more, 60% by mass or more, 70% by mass or more, or 80% by mass or more based on the total amount of the fibroin solution. The mass ratio of the content of the first solvent to the content of the second solvent (content of the first solvent/content of the second solvent) is such that the fibroin is suitably dissolved in the solvent and the tensile strength is increased. From the viewpoint of obtaining excellent fibroin fibers, the ratio may be, for example, 95/5 or more, 96/4 or more, 97/3 or more, or 98/2 or more. The mass ratio may be 99/1 or less.

The fibroin solution in this embodiment contains a first solvent with a low boiling point and a second solvent with a high boiling point, and thus, by performing dry spinning using this fibroin solution, a fiber (raw fiber) with excellent tensile strength can be obtained. In addition, since this raw fiber can be stretched in water, its strength can be further improved. Although the reason is unclear, the inventors speculate as follows.
That is, in order to improve the tensile strength by the secondary stretching, it is necessary to plastically deform the fibroin molecules to increase the rearrangement (orientation) of the molecular chains in the fiber axis. Water acts as a plasticizer for fibroin, and while it promotes molecular orientation during stretching, it is expected to have the effect of not excessively promoting crystallization. When stretching in water, if the crystallinity of the regenerated fibroin fiber is low, the penetration amount of water acting as a plasticizer is too large, and it becomes easy to break (melt-break) due to swelling, and conversely, if the crystallinity is too high, the strong cohesive force of fibroin prevents plastic deformation and makes it difficult to rearrange the molecular chains. In dry spinning using only the first solvent, only regenerated fibers with low crystallinity are obtained, so they melt-break in water. In contrast, if a second solvent with a boiling point higher than that of the first solvent is included, the second solvent remaining in the fiber acts as a plasticizer, bringing about the mobility of the fibroin molecules, promoting crystallinity, and allowing the secondary stretching to be performed in water without melt-breaking. Therefore, by using the second solvent in addition to the first solvent, it is possible to produce fibroin fibers that have appropriate crystallinity and excellent tensile strength, and it is also possible to draw them in water, thereby obtaining fibroin fibers with even greater strength.

Fibroin may be, for example, one or more types selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin. In particular, the fibroin may be silk fibroin, spider silk fibroin or a combination thereof. When silk fibroin and spider silk fibroin are used together, the proportion of silk fibroin may be, for example, 40 parts by mass or less, 30 parts by mass or less, or 10 parts by mass or less with respect to 100 parts by mass of spider silk fibroin.

Silk thread is a fiber (cocoon thread) obtained from the cocoons produced by silkworms, which are the larvae of Bombyx mori. Generally, one cocoon thread is composed of two silk fibroin threads and glue (sericin) covering them from the outside. Silk fibroin is composed of numerous fibrils. Silk fibroin is covered with four layers of sericin. Practically speaking, silk filaments obtained by dissolving and removing the outer sericin through refining are used for clothing. A typical silk thread has a specific gravity of 1.33, an average fineness of 3.3 decitex, and a fiber length of about 1300 to 1500 m. Silk fibroin can be obtained from natural or domestic silkworm cocoons, or from used or discarded silk fabrics.

The silk fibroin may be sericin-removed silk fibroin, sericin-unremoved silk fibroin, or a combination of these. Sericin-removed silk fibroin is purified by removing the sericin that covers the silk fibroin and other fats. Silk fibroin purified in this manner is preferably used as a freeze-dried powder. Sericin-unremoved silk fibroin is unpurified silk fibroin from which sericin and other substances have not been removed.

Spider silk fibroin may contain a spider silk polypeptide selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins (artificial spider silk proteins).

Natural spider silk proteins include, for example, large retractor dragline protein, weft protein, and phial gland protein. The large spinning tube bookmark thread has a repeating region consisting of a crystalline region and an amorphous region (also referred to as an amorphous region), so it has both high stress and elasticity. The weft of spider silk is characterized by having no crystalline regions but repeating regions consisting of amorphous regions. The weft threads have lower stress than the large spinning pipe drag threads, but have high elasticity.

The large spindle dragline protein is produced in the large fascia of spiders and is characterized by excellent toughness. Examples of the large spindle dragline protein include the large flial spidroins MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Araneus diadematus. ADF3 is one of the two major dragline proteins of the Japanese spider. Polypeptides derived from natural spider silk proteins may be polypeptides derived from these dragline silk proteins. Polypeptides derived from ADF3 are relatively easy to synthesize and have excellent properties in terms of strength and elongation and toughness.

Weft protein is produced in the flagelliform gland of spiders. Examples of weft proteins include flagelliform silk protein derived from Nephila clavipes.

The fibroin may be a modified fibroin (recombinant fibroin). The modified 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. The modified fibroin may further have amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but are not limited to, regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of about 100 amino acid residues.

As used herein, "modified fibroin" may be fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be fibroin whose domain sequence is the same as the amino acid sequence of naturally occurring fibroin. . "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. It is a protein that contains a domain sequence that is

A "modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as is, one that has had its amino acid sequence modified based on the amino acid sequence of naturally occurring fibroin (for example, one that has had its amino acid sequence modified by modifying the gene sequence of a cloned naturally occurring fibroin), or one that has been artificially designed and synthesized without relying on naturally occurring fibroin (for example, one that has a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence).

In this specification, the term "domain sequence" refers to the crystalline region (typically, corresponding to the (A) _n motif of the amino acid sequence) unique to fibroin and the amorphous region (typically, corresponding to the REP of the amino acid sequence). ), and is an amino acid sequence 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 consisting mainly of alanine residues, and the number of amino acid residues is 2 to 27. (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. . In addition, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) _n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A) _n motifs present in a plurality 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 10-200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences.

Modified fibroin can be obtained by modifying the amino acid sequence by, for example, substituting, deleting, inserting, and/or adding one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. You can get it at Substitutions, deletions, insertions and/or additions of amino acid residues can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.

Naturally derived 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. Specific examples include fibroin produced by insects or arachnids.

Examples of fibroin produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, and Eriogyna pyretor. um), Pilosamia Cynthia ricini ), Samia cynthia, Caligura japonica, Chussar silkworm (Antheraea mylitta), Muga silkworm (Antheraea assama), and silk proteins produced by silkworms such as Vespa si millima xanthoptera) larvae expel Examples include hornet silk protein.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank accession numbers M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

Fibroin produced by spiders includes, for example, spiders belonging to the Araneus genus (Araneus genus) such as Araneus spider, Japanese Araneus spider, Red Araneus spider, Blue Araneus spider, and Bean Araneus spider, and Araneus genus Araneus (Araneus genus) such as Araneus Araneus, Araneus Araneus, Araneus Araneus, Brown Araneus spider, and Satsuma flea spider. spiders that belong to the genus Cyrtarachne, such as spiders belonging to the genus Cyrtarachne, such as spiders belonging to the genus Pronus, such as the genus Pronus, spiders that belong to the genus Cyrtarachne, such as the genus Cyrtachne, and genus spiny spiders such as the spiny spider and the staghorn spider. Spiders belonging to the genus Ordgarius (genus Gasteracantha), spiders belonging to the genus Ordgarius (genus Ordgarius) such as the Japanese red-winged spider and spider spider, spiders belonging to the genus Argiope (genus Argiope) such as the Argiope spider, Argiope spider, etc. spiders belonging to the genus Arachnura (genus Arachnura); spiders belonging to the genus Acusilas (genus Acusilas), such as the spider spider; Spider silk protein produced by spiders belonging to the genus Cyclosa (genus Cyclosa), such as spiders belonging to the genus Chorizopes, such as spiders belonging to the genus Cyclosa, such as spiders belonging to the genus Chorizopes, and spiders belonging to the genus Chorizopes, such as the spider spider Spiders that belong to the genus Tetragnatha, such as the white-legged spider, the black-legged spider, and the scaly spider; spiders that belong to the genus Leucauge, such as the giant white-backed spider, the white-faced spider, and the white-faced spider; Spiders belonging to the genus Nephila (genus Nephila), spiders belonging to the genus Menosira (genus Menosira) such as the golden spider, spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the brown recluse spider, black widow spiders, redback spiders, brown widow spiders, brown widow spiders, etc. Examples include spider silk proteins produced by spiders belonging to the Tetragnathidae family, such as spiders belonging to the genus Latrodectus and spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include drag thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of spider silk proteins produced by arachnids include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] ] (GenBank accession number AAC04504 (amino acid sequence ), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein n spidroin 2 [derived from Nephila clavata] (GenBank accession No. AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major amplate spidroin 1 [derived from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and ma jor ampullate spidroin 2 [Euprosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor amp ullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), etc. Can be mentioned.

More specific examples of naturally derived fibroin include fibroins whose sequence information is registered in NCBI GenBank. For example, from among the sequences registered in NCBI GenBank that contain INV as a division, it can be confirmed by extracting sequences in which spidroin, amplify, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION, a specific product character string from CDS, and a specific character string in TISSUE TYPE from SOURCE.

The modified fibroin may be modified silk fibroin (the amino acid sequence of silk protein produced by silkworms has been modified), or modified spider silk fibroin (the amino acid sequence of spider silk protein produced by arachnids has been modified). It may be a thing). As the modified fibroin, modified spider silk fibroin is preferred.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large retractor dragline protein produced in the large flial gland of spiders, and a domain sequence with a reduced content of glycine residues. a modified fibroin having a domain sequence with a reduced content of (A) _n motif (a third modified fibroin), a content of glycine residues, and (A) _n Modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin with a domain sequence including a region with a locally large hydrophobicity index (fifth modified fibroin), and content of glutamine residues 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 in the (A) _n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, even more preferably an integer of 10 to 20, even more preferably an integer of 4 to 16, particularly preferably an integer of 8 to 16, and most preferably an integer of 10 to 16. 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, even more preferably 20 to 100 residues, and even more preferably 20 to 75 residues. The first modified fibroin is preferably such that the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by formula 1: [(A) _n motif-REP] _m is 40% or more, more preferably 60% or more, and even more preferably 70% or more, of the total number of amino acid residues.

The first modified fibroin contains an amino acid sequence unit represented by formula 1: [(A) _n motif-REP] _m , and has an amino acid sequence whose C-terminal sequence is shown in any of SEQ ID NOs: 1 to 3, or It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any 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 the C-terminal 50 amino acid residues of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is the same as that of ADF3 (GI: 1263287, NCBI). The amino acid sequence shown in SEQ ID NO: 3 is the same as that obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 1 is obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1. Identical to the amino acid sequence.

As a more specific example of the first modified fibroin, (1-i) the amino acid sequence shown by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence shown by SEQ ID NO: 4 and 90 Mention may be made of modified fibroin comprising amino acid sequences having % or more sequence identity. Preferably, the sequence identity is 95% or more.

The amino acid sequence shown by SEQ ID NO: 4 is the amino acid sequence of ADF3 with an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus. The 13th repeat region has been increased approximately twice as much, and the translation has been mutated so that it terminates at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown by SEQ ID NO: 4 is the same as the amino acid sequence shown by SEQ ID NO: 3.

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

The second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues compared to naturally occurring fibroin. The second modified fibroin can be said to have an amino acid sequence that corresponds to at least one or more glycine residues in REP being substituted with another amino acid residue, compared to 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), compared to naturally-derived fibroin. ) has an amino acid sequence corresponding to the substitution of one glycine residue in at least one or more of the motif sequences with another amino acid residue. You can.

In the second modified fibroin, the proportion of the motif sequence in which the above-mentioned glycine residue is substituted with another amino acid residue may be 10% or more of the total motif sequence.

The second modified fibroin includes a domain sequence represented by formula 1: [(A) _n motif-REP] _m , and from the above domain sequence, from the (A) _n motif located at the most C-terminal side to the above domain sequence. Let z be the total number of amino acid residues in the amino acid sequence consisting of z/w is 30% or more, where w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the above domain sequence, 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 95% or more. It is more preferable that it be present, and even more preferable that it be 100% (meaning that it is composed only of alanine residues).

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

The method for calculating z/w will be explained in more detail. First, in fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , (A) _n is located closest to the C-terminus from the domain sequence. Amino acid sequences consisting of XGX are extracted from all REPs included in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, if 50 amino acid sequences consisting of XGX are extracted (no overlap), z is 50×3=150. In addition, for example, in the case of an amino acid sequence consisting of XGXGX, if there is an X included in two XGX (center be). w is the total number of amino acid residues contained in the domain sequence excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of 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 (excluding the (A) _n motif located on the most C-terminal side). Next, by dividing z by w, z/w (%) can be calculated.

Here, z/w in naturally derived fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified, and 663 types of fibroin (of which, 415 types of fibroin derived from spiders) were extracted. Among all the extracted fibroins, z/w was calculated from the amino acid sequence of naturally derived fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , in which the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less, by the above-mentioned calculation method. The results are shown in FIG. 2. The horizontal axis of FIG. 2 indicates z/w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, z/w in all naturally derived fibroins is less than 50.9% (the highest is 50.86%).

In the second modified fibroin, z/w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, and 70% or more. Even more preferably, it is 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 can be produced, for example, by modifying the cloned naturally occurring fibroin gene sequence so that it encodes another amino acid residue by replacing at least a portion of the base sequence encoding a glycine residue. Obtainable. At this time, one glycine residue in the GGX motif and GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z/w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence that satisfies the above aspects from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification equivalent to replacing the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally-derived fibroin, one or more amino acid residues are further substituted or deleted. , insertion and/or addition may be made to the amino acid sequence.

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

More specific examples of the second modified fibroin include (2-i) modified fibroins containing an amino acid sequence shown in 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 shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

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

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

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

The modified fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin (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 (2-ii) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has XGX ( However, when z is the total number of amino acid residues in the amino acid sequence consisting of is preferably 50.9% or more.

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

Examples of tag sequences include affinity tags that utilize specific affinity (binding properties, affinity) with other molecules. A specific example of the affinity tag is a histidine tag (His tag). His tag is a short peptide with about 4 to 10 histidine residues arranged in a row, and has the property of specifically binding to metal ions such as nickel, making it possible to isolate modified fibroin by chelating metal chromatography. It can be used for. A specific example of the tag sequence is, for example, the amino acid sequence shown by SEQ ID NO: 11 (an amino acid sequence including a His tag sequence and a hinge sequence).

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

Furthermore, "epitope tags" that utilize antigen-antibody reactions can also be used. By adding a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag, and the like. By using epitope tags, modified fibroin can be easily purified with high specificity.

Furthermore, it is also possible to use tag sequences whose tag sequences can be cleaved with a specific protease. Modified fibroin from which the tag sequence has been removed can also be recovered by treating the protein adsorbed via the tag sequence with a protease.

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

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

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

The modified fibroin (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin (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 (2-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and has XGX ( However, when z is the total number of amino acid residues in the amino acid sequence consisting of is preferably 50.9% or more.

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

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

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

The third modified fibroin has a domain sequence that is at least one (A) _n motif for every one to three (A) _n motifs from the N-terminal side to the C-terminal side, compared to naturally-derived fibroin. may have an amino acid sequence corresponding to the deletion.

The third modified fibroin may have an amino acid sequence in which the domain sequence corresponds to at least two consecutive (A) _n motifs deleted from the N-terminus to the C-terminus, and one (A) _n motif deleted in this order, compared to naturally occurring fibroin.

The third modified fibroin may have an amino acid sequence whose domain sequence corresponds to deletion of (A) _n motif from every second domain sequence from the N-terminal side to the C-terminal side. .

The third modified fibroin contains a domain sequence represented by formula 1: [(A) _n motif-REP] _m , and two adjacent [(A) _n motifs from the N-terminal side toward the C-terminal side. -REP] sequentially compare the number of amino acid residues of the REPs in the unit, and when the number of amino acid residues of the REP with the smallest number of amino acid residues is set as 1, the ratio of the number of amino acid residues of the other REP is 1.8 ~ 11.3, where x is the maximum value of the sum of the numbers of amino acid residues in two adjacent [(A) _n motif-REP] units, and y is the total number of amino acid residues in the domain sequence. Furthermore, it may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is more preferable that it be present, and even more preferable that it be 100% (meaning that it is composed only of alanine residues).

The method for calculating x/y will be explained in more detail with reference to FIG. FIG. 1 shows the domain sequence of modified fibroin with the N-terminal sequence and C-terminal sequence removed. The domain sequence is from the N-terminal side (left side): (A) _n motif - 1st REP (50 amino acid residues) - (A) _n motif - 2nd REP (100 amino acid residues) - (A) _n Motif - 3rd REP (10 amino acid residues) - (A) _n motif - 4th REP (20 amino acid residues) - (A) _n motif - 5th REP (30 amino acid residues) - (A) It has a sequence called _n motif.

Two adjacent [(A) _n- motif-REP] units are selected sequentially from the N-terminus to the C-terminus 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 first REP and second REP, and comparison of third REP and fourth REP), pattern 2 (comparison of first REP and second REP, and Pattern 3 (Comparison of the 2nd REP and the 3rd REP, and Comparison of the 4th REP and the 5th REP), Pattern 4 (Comparison of the 1st REP and the 5th REP) A comparison of the second REP) was shown. Note that there are other selection methods besides this.

Next, for each pattern, the number of amino acid residues of each REP in the two selected adjacent [(A) _n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of amino acid residues of the other amino acid residue, when the one with the smaller number of 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), if the first REP with fewer amino acid residues is set as 1, then the second REP 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 with fewer amino acid residues is set as 1, the fifth REP The ratio of the number of amino acid residues is 30/20=1.5.

In Figure 1, a pair of [(A) _n -motif-REP] units in which the ratio of the number of amino acid residues in the other is 1.8 to 11.3 when the number of amino acid residues in the other is set to 1 is shown by a solid line. In this specification, this ratio is referred to as the "jaggy ratio." A pair of [(A) _n -motif-REP] units in which the ratio of the number of amino acid residues in the other is less than 1.8 or more than 11.3 when the number of amino acid residues in the other is set to 1 is shown by a dashed line.

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

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

In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, and even more preferably 70% or more. It is preferably at least 75%, even more preferably at least 80%, 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 serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x/y is preferably 89.6% or more. /y is preferably 77.1% or more, and when the jagged ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the jagged ratio is 1:1.9 to 8.4. : 1.9 to 4.1, x/y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which at least seven of the (A) _n motifs present in the domain sequence are composed of only alanine residues, x/y is preferably 46.4% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more. There is no particular upper limit to x/y, and it is sufficient that it is 100% or less.

Here, x/y in naturally derived fibroin will be explained. First, as described above, when fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method described above, 663 types of fibroin (415 types of fibroin derived from arachnids) were extracted. Of all the extracted fibroins, from the amino acid sequence of naturally occurring fibroin consisting of the domain sequence represented by formula 1: [(A) _n motif-REP] _m , x/y was calculated. Figure 3 shows the results when the jaggedness ratio is 1:1.9 to 4.1.

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

The third modified fibroin can be obtained, for example, by deleting one or more of the sequences encoding the (A) _n motif from the gene sequence of the cloned naturally-occurring fibroin so that x/y is 64.2% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence corresponding to the deletion of one or more (A) _n motifs from the amino acid sequence of the naturally-occurring fibroin so that x/y is 64.2% or more, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of the naturally-occurring fibroin, the amino acid sequence may be modified by further substituting, deleting, inserting and/or adding one or more amino acid residues.

As a more specific example 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 -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.

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

The x/y value of the amino acid sequence shown by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) at a jagged ratio of 1:1.8 to 11.3 is 15.0%. The value of x/y in both the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 is 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%. The values of z/w in the amino acid sequences shown 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%, and 66. 1% and 70.0%.

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

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

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

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

The amino acid sequences shown by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are the amino acid sequences shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in (including His tag sequence and hinge sequence) is added.

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

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

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

The fourth modified fibroin has a domain sequence that has a reduced content of (A) _n motifs as well as an amino acid sequence that has a reduced content of glycine residues compared to naturally-derived fibroin. It is something that you have. The fourth modified fibroin has a domain sequence in which at least one or more (A) _n motifs are deleted compared to naturally occurring fibroin, and in addition, at least one or more glycine residues in REP are deleted. It can be said to have an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin that has both the characteristics of the above-described second modified fibroin and the third modified fibroin. Specific aspects are as explained in the second modified fibroin and the third modified fibroin.

More specific examples of the fourth modified fibroin include (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), and SEQ ID NO: 13 (PRT410). ), the amino acid sequence shown 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 Examples of modified fibroin include an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in . Specific embodiments of the modified fibroin comprising the amino acid sequence shown 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 are substituted with amino acid residues having a large hydrophobicity index compared to naturally occurring fibroin, and/or REP It may have an amino acid sequence that includes a region with a locally large hydrophobic index, which corresponds to the insertion of one or more amino acid residues with a large hydrophobic index.

Preferably, a region with a locally large hydrophobicity index is composed of 2 to 4 consecutive amino acid residues.

The above-mentioned amino acid residues having a large hydrophobicity index are amino acids 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, in addition to modifications corresponding to the replacement of one or more amino acid residues in REP with amino acid residues having a high hydrophobicity index and/or the insertion of one or more amino acid residues having a high hydrophobicity index into REP, modifications in the amino acid sequence corresponding to the replacement, deletion, insertion and/or addition of one or more amino acid residues compared to naturally occurring fibroin.

For example, the fifth modified fibroin can be obtained by replacing one or more hydrophilic amino acid residues (for example, amino acid residues with a negative hydrophobicity index) in REP with hydrophobic amino acid residues from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting a group (for example, an amino acid residue with a positive hydrophobic index) and/or by inserting one or more hydrophobic amino acid residues into REP. In addition, for example, one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally-derived fibroin, and/or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to the insertion of , and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acids in REP In addition to the modification corresponding to the insertion of a residue, the amino acid sequence may be further modified corresponding to the substitution, deletion, insertion, and/or addition of one or more amino acid residues.

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

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

The method for calculating p/q will be explained in more detail. For calculation, use formula 1: [(A) _n motif - REP] From the domain sequence represented by _m , remove the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of the domain sequence. (hereinafter referred to as "array A") is used. 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 sum of HI of each amino acid residue included 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 to calculate the average value 1 to 4 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 if a certain amino acid residue corresponds to multiple "four consecutive amino acid residues with an average value of hydrophobicity index of 2.6 or more", it may be included as one amino acid residue in the region. become. The total number of amino acid residues included in the region is p. Further, the total number of amino acid residues included in sequence A is q.

For example, if 20 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" are extracted (no overlap), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2. The region with a value of .6 or more includes 20 consecutive 4 amino acid residues (no overlap), and p is 20×4=80. Also, for example, if two "four consecutive amino acid residues with an average value of hydrophobicity index of 2.6 or more" overlap by one amino acid residue, the hydrophobicity index of the four consecutive amino acid residues The region where the average value of is 2.6 or more will contain 7 amino acid residues (p=2×4−1=7. “−1” is the deduction of overlap.). For example, in the case of the domain sequence shown in Figure 4, there are 7 non-overlapping "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", so p is 7 x 4 = It becomes 28. Further, 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 (not including the (A) _n motif present at the end of the C-terminal side). Next, p/q (%) can be calculated by dividing p by q. In the case of FIG. 4, it is 28/170=16.47%.

In the fifth modified fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, and even more preferably 20% or more. It is even more preferable, and even more preferable that it is 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 more hydrophilic amino acid residues in REP (e.g., hydrophobic index substituting a hydrophobic amino acid residue (e.g., an amino acid residue with a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid residues into the REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region with a large hydrophobicity index. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above p/q condition from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more amino acid residues in REP have been substituted with amino acid residues with a higher hydrophobic index compared to naturally occurring fibroin, and/or one or more amino acid residues in REP In addition to the modification that corresponds to the insertion of an amino acid residue with a large hydrophobicity index, modifications that correspond to the substitution, deletion, insertion, and/or addition of one or more amino acid residues may also be performed. .

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

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

The modified fibroin (5-i) will be explained. The amino acid sequence shown by SEQ ID NO: 19 is different from the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410), except for the terminal domain sequence on the C-terminal side, consisting of 3 amino acid residues in every other REP. The amino acid sequence (VLI) is inserted at two positions, some glutamine (Q) residues are replaced with serine (S) residues, and some amino acids on the C-terminal side are deleted. The amino acid sequence shown by SEQ ID NO: 20 is obtained by inserting an amino acid sequence (VLI) consisting of 3 amino acid residues at every other REP in the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525). be. The amino acid sequence shown by SEQ ID NO: 21 is obtained by inserting an amino acid sequence (VLI) consisting of 3 amino acid residues at two positions in every other REP with respect to the amino acid sequence shown by SEQ ID NO: 8.

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

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

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

More specific examples of modified fibroin containing a tag sequence include (5-iii) the amino acid sequence shown by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666); or (5-iv) ) Modified fibroin containing 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 can be mentioned.

The amino acid sequences shown by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 have the amino acid sequence shown by SEQ ID NO: 11 (His tag) at the N-terminus of the amino acid sequences shown by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (including the hinge arrangement and the hinge arrangement).

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

The modified fibroin (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 (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 (5-iv) has 90% or more sequence identity with the amino acid sequence shown 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 Amino acids included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs included in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence. When the total number of residues is p, and the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif to the C-terminus of the domain sequence from the domain sequence located on the most C-terminal side is q. , p/q is preferably 6.2% or more.

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

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

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

When the sixth modified fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is 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 this specification, the "GPGXX motif content" is a value calculated by the following method.
In a fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by formula 1: [(A _{) n} motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif, the GPGXX motif content is calculated as s/t, where s is three times the total number of GPGXX motifs contained in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence (i.e., equivalent to the total number of G and P in the GPGXX motif), and t is the total number of amino acid residues in all REPs obtained by excluding from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence and further excluding the (A) _n motif.

In calculating the GPGXX motif content rate, the target is the "sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence". (A) The sequence from _{the n} motif to the C-terminus of the domain sequence (corresponding to REP) may include sequences that have low correlation with sequences characteristic of fibroin, and m is small. (that is, when the domain sequence is short), this will affect the calculation result of the GPGXX motif content, so this is to eliminate this influence. Note that when a "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA".

FIG. 5 is a schematic diagram showing the domain sequence of modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 5. First, in the domain sequence of the modified fibroin shown in Figure 5 (which is of the "[(A) _n motif-REP] _m - (A) _n motif" type), all REPs are "located at the most C-terminal side. (A) A sequence obtained by excluding the sequence from _{the n} motif to the C-terminus of the domain sequence from the domain sequence" (sequence shown as "region A" in Figure 5), so it is necessary to calculate s. The number of GPGXX motifs is 7, and s is 7×3=21. Similarly, all REPs are defined as "a sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence" (the sequence indicated by "Region A" in Figure 5). ), the total number t of amino acid residues in all REPs, excluding the (A) _n motif, is 50+40+10+20+30=150. Next, s/t (%) can be calculated by dividing s by t, which is 21/150=14.0% for the modified fibroin in FIG.

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

In this specification, "glutamine residue content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - ( _A ) fibroin (modified fibroin or naturally In fibroin), everything contained in the sequence (corresponding to "region A" in Figure 5) obtained by excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to "region A" in Figure 5). In REP, the total number of glutamine residues included in that region is u, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and then (A) _n The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues in all REPs excluding motifs. In calculating the glutamine residue content, we target "sequences obtained by excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of the domain sequence from the domain sequence" due to the above-mentioned reasons and The same is true.

The sixth modified fibroin corresponds to a domain sequence in which one or more glutamine residues in REP are deleted or substituted with other amino acid residues compared to naturally occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be any amino acid residue other than glutamine residue, but is preferably an amino acid residue with a higher hydrophobicity index than glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

As shown in Table 1, amino acid residues with a higher hydrophobic 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) may be mentioned. can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

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

In this specification, "hydrophobicity of REP" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - ( _A ) fibroin (modified fibroin or naturally In fibroin), everything contained in the sequence (corresponding to "region A" in Figure 5) obtained by excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to "region A" in Figure 5). In REP, the sum of the hydrophobicity indices of each amino acid residue in that region is v, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues in all REPs excluding the _n motif. In calculating the degree of hydrophobicity of REP, we target "sequences obtained by excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of the domain sequence from the domain sequence" due to the above-mentioned reasons and The same is true.

The sixth modified fibroin has a domain sequence in which one or more glutamine residues in REP are deleted compared to naturally occurring fibroin, and/or one or more glutamine residues in REP In addition to modifications corresponding to the substitution of other amino acid residues, there may also be modifications of the amino acid sequence corresponding to substitutions, deletions, insertions, and/or additions of one or more amino acid residues. .

The sixth modified fibroin can be produced by, for example, deleting one or more glutamine residues in REP from the cloned naturally-derived fibroin gene sequence, and/or replacing one or more glutamine residues in REP with another one. can be obtained by substituting the amino acid residue of In addition, for example, one or more glutamine residues in REP have been deleted from the amino acid sequence of naturally-derived fibroin, and/or one or more glutamine residues in REP have been replaced with other amino acid residues. It can also be obtained by designing a corresponding amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

More specific examples of the sixth modified fibroin include (6-i) modified fibroin containing the amino acid sequence shown in SEQ ID NO:25 (Met-PRT888), SEQ ID NO:26 (Met-PRT965), SEQ ID NO:27 (Met-PRT889), SEQ ID NO:28 (Met-PRT916), SEQ ID NO:29 (Met-PRT918), SEQ ID NO:30 (Met-PRT699), SEQ ID NO:31 (Met-PRT698) or SEQ ID NO:32 (Met-PRT966), or (6-ii) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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 or SEQ ID NO:32.

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

The amino acid sequence shown in SEQ ID NO:30 is the amino acid sequence shown in SEQ ID NO:8 (Met-PRT525) in which all QQs have been replaced with VL. The amino acid sequence shown in SEQ ID NO:31 is the amino acid sequence shown in SEQ ID NO:8 in which all QQs have been replaced with VL and the remaining Qs have been replaced with I.

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

The amino acid sequences shown 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 and SEQ ID NO: 32 all have a glutamine residue content of 9% or less. Yes (Table 2).

The modified fibroin (6-i) may consist of the amino acid sequence shown in 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, or SEQ ID NO:32.

The modified fibroin of (6-ii) has at least 90% the amino acid sequence shown 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 or SEQ ID NO: 32. It contains an amino acid sequence having sequence identity. 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. It is a protein that contains a sequence. The sequence identity is preferably 95% or more.

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

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

More specific examples of modified fibroin containing tag sequences include (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), modified fibroin comprising the amino acid sequence shown by SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698) or SEQ ID NO: 40 (PRT966), or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: Examples include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

The amino acid sequences shown 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, and SEQ ID NO: 40 are SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively. , SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, and the amino acid sequence shown by SEQ ID NO: 11 (including the His tag sequence and hinge sequence) was added to the N-terminus of the amino acid sequence shown by SEQ ID NO: 31 and SEQ ID NO: 32. It is something. Since the tag sequence was only 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 Both of the amino acid sequences shown by SEQ ID NO: 40 have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) consists of the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. There may be.

The modified fibroin of (6-iv) has at least 90% the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. It contains an amino acid sequence having sequence identity. 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. It is a protein that contains a sequence. The sequence identity is preferably 95% or more.

The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less. The modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

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

The modified fibroin has at least two or more of the characteristics possessed by 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. It may also be a modified fibroin that has the following characteristics.

<Method for producing modified fibroin>
The modified fibroin according to any of the above embodiments can be used, for example, in 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.

The method for producing a nucleic acid encoding a modified fibroin is not particularly limited. For example, the nucleic acid can be produced by a method in which a gene encoding a natural fibroin is amplified and cloned by polymerase chain reaction (PCR) or the like, and modified by genetic engineering techniques, or by a chemical synthesis method. The method for chemically synthesizing a nucleic acid is also not particularly limited. For example, a gene can be chemically synthesized by a method in which oligonucleotides automatically synthesized using AKTA oligopilot plus 10/100 (GE Healthcare Japan, Ltd.) or the like are linked by PCR or the like based on amino acid sequence information of fibroin obtained from the NCBI web database or the like. At this time, in order to facilitate purification and/or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of an initiation codon and a His10 tag is added to the N-terminus of the above amino acid sequence may be synthesized.

The regulatory sequence is a sequence (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) that controls the expression of modified fibroin in the host, and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce expression of the modified fibroin may be used. An inducible promoter is a promoter whose transcription can be controlled 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.

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, or an artificial chromosome vector. As the expression vector, one that is capable of autonomous replication in the host cell or can be integrated into the chromosome of the host, and contains a promoter at a position where the nucleic acid encoding the modified fibroin can be transcribed is preferably 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.

Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphyllum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of microorganisms 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, vectors for introducing a nucleic acid encoding modified fibroin include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, Examples include pNCO2 (Japanese Patent Application Laid-open No. 2002-238569).

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.). Examples of yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces, and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

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

In addition to direct expression, methods for expressing nucleic acids by hosts transformed with expression vectors include secretory production, fusion protein expression, etc. according to the methods described in Molecular Cloning, 2nd edition. .

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 it from the culture medium. The host can be cultured in a culture medium according to a method commonly used for culturing hosts.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and allows efficient culture of the host. Either a natural medium or a synthetic medium may be used.

The carbon source may be anything that can be assimilated by the transformed microorganisms, such as carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch and starch hydrolysates, and acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used. Examples of nitrogen sources include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermented microbial cells, and digested products thereof can be used. As the inorganic salts, for example, potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions, for example, by shaking culture or deep aeration agitation culture. The culture temperature is, for example, 15 to 40°C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

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

The expressed modified fibroin can be isolated and purified by commonly used methods. For example, when the modified fibroin is expressed in a dissolved state in cells, after the completion of culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then processed using an ultrasonicator, French press, or Manton. Host cells are disrupted using a Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, methods commonly used for isolation and purification of proteins, such as solvent extraction, salting out with ammonium sulfate, desalting, and organic solvents. precipitation method, diethylaminoethyl (DEAE)-Sepharose, anion exchange chromatography method using resins such as DIAION HPA-75 (manufactured by Mitsubishi Kasei Corporation), and positive chromatography method using resins such as S-Sepharose FF (manufactured by Pharmacia Corporation). Ion exchange chromatography, hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing. A purified sample can be obtained by using these methods alone or in combination.

In addition, when the modified fibroin is expressed by forming an insoluble body within the cells, the host cells are similarly collected, disrupted, and centrifuged to recover the insoluble body of the modified fibroin as a precipitate fraction. The recovered insoluble modified fibroin can be solubilized with a protein denaturing agent. After this operation, a purified sample of modified fibroin can be obtained by the same isolation and purification method as 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 processing a culture using techniques 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.

The content of fibroin may be 3% by mass or more, 5% by mass or more, 10% by mass or more, 15% by mass or more, or 20% by mass or more, and 40% by mass or less, 35% by mass or more based on the total amount of the fibroin solution. % or less, 30% by mass or less, or 25% by mass or less.

The viscosity of the fibroin solution may be appropriately set depending on the intended use. When using a fibroin solution as a spinning solution, it is not particularly limited as long as it has a viscosity that allows dry spinning, but from the viewpoint of productivity, the viscosity of the fibroin solution is, for example, 10,000 mPa·sec at 25°C. or more, 200,000 mPa・sec or less, 150,000 mPa・sec or less, 100,000 mPa・sec or less, 80,000 mPa・sec or less, 50,000 mPa・sec or less, 30,000 mPa・sec or less, 20, 000 mPa·sec or less, or 15,000 mPa·sec or less. The viscosity of the fibroin solution can be measured using, for example, "EMS Viscometer" (trade name, manufactured by Kyoto Electronics Industry Co., Ltd.).

The fibroin solution may further contain various additives as necessary. Examples of additives include plasticizers, leveling agents, crosslinking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, and synthetic resins. The content of the additive may be 50% by mass or less based on the total amount of the fibroin solution.

The fibroin solution described above is suitable as a solution (dope solution) used for producing fibers containing fibroin (fibroin fibers), and is used for spinning (especially preferably dry spinning) of fibers containing fibroin (fibroin fibers). It is particularly suitable as a solution (dope solution or spinning solution).

[Method of producing fibroin fiber]
The method for producing a fibroin fiber according to this embodiment includes a step of spinning the above-mentioned fibroin solution (dope solution or spinning solution) by dry spinning (spinning step).

In the spinning process, the spinning solution is discharged from a spinneret into the air, and then heated to evaporate the solvent to obtain fibroin fibers (coagulated threads). The spinning process can be carried out by a known dry spinning method, for example, except that the above-mentioned fibroin solution is used as the spinning solution. The spinning process can also be carried out by electrospinning (electrostatic spinning), but from the viewpoint of simplifying the manufacturing process and reducing manufacturing costs, it is preferable to carry out the spinning process by a method other than electrospinning.

Although there are no particular limitations on the method for discharging the fibroin solution (spinning solution) from the spinneret, for example, a method using a metering pump as a means for feeding the fibroin solution (spinning solution) can be used. The discharge amount can be adjusted as appropriate depending on the production speed.

The temperature of the fibroin solution (spinning solution) when passing through the spinneret and the temperature of the spinneret are not particularly limited, and may be adjusted as appropriate depending on the concentration and viscosity of the fibroin solution used, the type of solvent, etc. The temperature is preferably 30°C to 100°C from the viewpoint of preventing deterioration of fibroin. The upper limit of the temperature is set to a temperature below the boiling point of the solvent in the fibroin solution, and the upper limit is set to a temperature below the boiling point of the solvent in the fibroin solution, from the viewpoint of reducing the possibility of pressure increase due to solvent volatilization and clogging in the piping due to solidification of the fibroin solution. It is preferable to set the above temperature as the lower limit. This improves process stability.

The shape of the spinneret, the shape of the holes, the number of holes, etc. are not particularly limited and can be appropriately selected according to the desired fiber diameter and the number of single yarns. For example, when the shape of the spinneret holes is circular, the hole diameter can be 0.01 mm or more and 0.6 mm or less. If the hole diameter is 0.01 mm or more, pressure loss can be reduced and equipment costs can be suppressed. If the hole diameter is 0.6 mm or less, the need for a stretching operation to narrow the fiber diameter can be reduced, and the possibility of stretching breakage occurring between discharge and withdrawal can be reduced.

The manufacturing method according to the present embodiment may further include a step of filtering the spinning solution before discharging the fibroin solution (filtration step) and/or a step of defoaming the spinning solution before discharging the fibroin solution (defoaming step). .

Fibroin fibers obtained by the spinning process may be used as they are for any purpose. Alternatively, a step of stretching the fibroin fibers (stretching step) may be further performed to obtain fibroin fibers stretched to an arbitrary stretching ratio, and the obtained fibroin fibers may be used for any purpose.

That is, the method for producing fibroin fibers may further include a step of drawing the spun fibroin fibers (drawing step) after the spinning step. Examples of the stretching method include wet heat stretching and dry heat stretching.

The stretching method is preferably moist heat stretching, and more preferably underwater stretching, in which fibroin fibers are stretched in water, from the viewpoint of obtaining fibroin fibers with even better strength. The temperature of water during underwater stretching may be 20°C or higher, 30°C or higher, 40°C or higher, 50°C or higher, or 60°C or higher, and may be 90°C or lower, 85°C or lower, or 80°C or lower. .

Dry heat stretching can be performed using equipment such as a contact hot plate or a non-contact furnace, but is not particularly limited. Any device that can stretch at a certain magnification may be used. The temperature may be, for example, 100°C to 270°C, 140°C to 230°C, 140°C to 200°C, 160°C to 200°C, 160°C to 180°C. It may be ℃.

The stretching ratio in the drawing step may be, for example, 1 to 30 times, 1 to 25 times, or 1 to 20 times the raw material fiber (undrawn yarn or pre-drawn yarn). , may be 1 to 15 times, may be 1 to 10 times, may be 2 to 10 times, may be 2 to 8 times, may be 2 to 6 times, may be 2 to 4 times It may be 2 to 3 times as large.

In the stretching step, wet heat stretching (underwater stretching) and dry heat stretching may be performed individually, or these may be performed in multiple stages or in combination. That is, as a stretching process, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and then the third stage stretching is performed by wet heat stretching. The stretching can be carried out by dry heat stretching, or by appropriately combining wet heat stretching and dry heat stretching.

The lower limit of the final draw ratio of the fibroin fibers after the drawing process is, for example, 1, 2, 3, 4, 5, 6 times the raw material fiber (undrawn yarn or pre-drawn yarn). It may be any one of 2 times, 7 times, 8 times, or 9 times. The upper limit of the final stretching ratio of the spider silk fibroin fiber after the stretching process is, for example, 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. It may be either. Further, for example, the final stretching ratio may be 3 to 40 times, may be 3 to 30 times, may be 5 to 30 times, may be 5 to 20 times, and may be 5 to 15 times. may be 5 to 13 times larger. However, the stretching ratio is not limited as long as the desired fiber thickness, mechanical properties, and other characteristics can be obtained.

Before or after the stretching process, an oil agent may be applied to the fibers, if necessary, for the purpose of imparting antistatic properties, convergence properties, lubricity, etc. The type of oil to be applied, the amount to be applied, etc. are not particularly limited, and can be adjusted as appropriate in consideration of the use of the fibers, the ease of handling of the fibers, and the like.

〔product〕
The product according to this embodiment may be various products containing the above-mentioned fibroin fibers. Products can include, for example, fibers, yarns, fabrics, knits, plaits, nonwovens, paper, and cotton. Examples of fibers include long fibers, short fibers, monofilaments, and multifilaments, and examples of yarns include spun yarns, twisted yarns, false twisted yarns, textured yarns, mixed yarns, and mixed yarns. I can do it. Further, from these fibers and threads, fabrics such as woven fabrics, knitted fabrics, braided fabrics, nonwoven fabrics, etc., paper, cotton, etc. can be manufactured. These products can be manufactured by known methods.

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

[Production of modified fibroin]
(1) Preparation of expression vector Based on the base sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), modified spider silk fibroin having SEQ ID NO: 40 ( (hereinafter also referred to as "PRT966"). The amino acid sequence shown by SEQ ID NO: 40 is the amino acid sequence derived from Nephila clavipes, whereas the amino acid sequence shown by SEQ ID NO: 40 is the amino acid sequence shown by SEQ ID NO: 7 for the purpose of improving hydrophobicity. It has a sequence in which the 20 domain sequence regions present in the sequence are repeated twice, all QQs in the sequence are replaced with VF, and the remaining Qs are replaced with I, and furthermore, the N-terminus has SEQ ID NO: 11. The indicated amino acid sequences (tag and hinge sequences) have been added.

Next, a nucleic acid encoding modified spider silk fibroin PRT966 having the designed amino acid sequence SEQ ID NO: 40 was synthesized. An NdeI site and an EcoRI site downstream of the stop codon were added to the nucleic acid at the 5' end. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Expression of modified fibroin Escherichia coli BLR (DE3) was transformed with the expression vector obtained in (1). The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium containing ampicillin (Table 4) so that the OD ₆₀₀ was 0.005. The culture solution temperature was kept at 30°C, and flask culture was performed until the OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) so that the OD ₆₀₀ was 0.05. The culture temperature was kept at 37° C., and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. Culture was carried out by keeping the culture solution temperature at 37° C. and controlling the pH to be constant at 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and culture was performed for 20 hours. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1mM to induce expression of the modified fibroin. After 20 hours had passed after the addition of IPTG, the culture solution was centrifuged and the bacterial cells were collected. SDS-PAGE was performed using the bacterial cells prepared from the culture medium before and after the addition of IPTG, and the appearance of a band of the desired modified fibroin size that depended on the addition of IPTG indicated the expression of the desired artificial structural protein. confirmed.

(3) Purification of modified fibroin The bacterial cells collected 2 hours after adding IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted using a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH 7.0) to a concentration of 100mg/mL and incubated at 60°C. The mixture was stirred with a stirrer for 30 minutes to dissolve the mixture. After dissolution, dialysis was performed against water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation, water was removed using a freeze dryer, and a freeze-dried powder was collected to obtain modified fibroin (PRT966).

[Preparation of fibroin solution (dope solution)]
<Examples 1-1 to 1-3>
The obtained modified fibroin was dissolved in a solvent to a concentration of 20% by mass to prepare a fibroin solution (dope solution). The solvent used was a mixture of hexafluoroisopropanol (HFIP, boiling point: 58°C) and formic acid (boiling point: 101°C) as a second component at the mass ratio shown in Table 6.

<Reference examples 1-1 to 1-3>
A fibroin solution (dope solution) was prepared in the same manner as in Examples 1-1 to 1-3, except that acetone (boiling point: 57° C.) was mixed as the second component at the mass ratio shown in Table 6.

<Comparative example 1>
A fibroin solution (dope solution) was prepared in the same manner as in Examples 1-1 to 1-3 except that only HFIP was used as the solvent.

[Preparation of fibroin fiber]
Each of the fibroin solutions of Examples 1-1 to 1-3, Reference Examples 1-1 to 1-3, and Comparative Example 1 was attached to the tip of a spinning syringe filled with the solution, with a diameter of 0.25 mm and a length of 12 mm. It was discharged through a 7 mm or 25.4 mm nozzle. The formed thread is wound at a speed of 1.0 m/min or 2.5 m/min by a first roller placed 50 cm directly below the nozzle, and then wound by a winding machine at the same speed via a second roller. I took it. The fibroin fibers thus produced were stored in a constant temperature and humidity environment (temperature 20±2° C., relative humidity 65±2%).

[Evaluation of fibroin fiber]
The tensile strength of the obtained fibroin fibers was measured using a tensile tester (INSTRON3342) at a relative humidity of 65%. The tensile strength was evaluated by a relative value when the tensile strength when only HFIP was used as a solvent (Comparative Example 1) was taken as 100. The relative values are shown in Table 6.

In addition, the obtained fibers were stretched on the same day or after being left in an environment of 20° C. and 65% RH for one day. In underwater stretching, a continuous stretching machine equipped with a delivery roller and a take-up roller is used, the water temperature during stretching is 20°C, the length of the fiber immersed in water is 30 cm, and the stretching ratio is 1 to 3 times. The conditions were set as follows. Immediately after stretching, the film was wound around a hot roller at 60° C. to dry, and then wound up using a winder. The suitability for underwater stretching was evaluated based on the following criteria. The results are shown in Table 6.
A: Underwater stretching was possible, and the fibers after stretching could be collected for 5 minutes or more.
B: Underwater stretching was possible and the fibers could be collected for less than 5 minutes after stretching.
C: Underwater stretching was possible, but the fibers could not be collected after stretching.
D: Underwater stretching was not possible.

As can be seen from Table 6, in the examples using a mixture of HFIP and a solvent with a boiling point higher than that of HFIP, in the comparative example using only HFIP, and in the comparative example using a mixture of HFIP with a solvent with a lower boiling point than HFIP. The tensile strength is significantly improved compared to the reference example. Furthermore, the examples are also excellent in suitability for underwater stretching.

Claims (9)

Dry spinning a dope solution containing fibroin and a solvent containing a first solvent as a main component and a second solvent having a boiling point higher than the boiling point of the first solvent as an additive component. A method for producing fibroin fibers, comprising: (except when the dry spinning is electrospinning). The method for producing fibroin fibers according to claim 1, wherein the mass ratio of the content of the first solvent to the content of the second solvent is 95/5 or more. The method for producing fibroin fibers according to claim 1 or 2, further comprising a step of stretching the fibers obtained by the above step in water. The fibroin fiber according to any one of claims 1 to 3, wherein the first solvent has a boiling point of 40°C or more and 120°C or less, and the second solvent has a boiling point of 80°C or more and 280°C or less. Production method. The method for producing fibroin fibers according to any one of claims 1 to 4, wherein the fibroin is derived from an insect or arachnid. The method for producing fibroin fibers according to any one of claims 1 to 5, wherein the fibroin is a modified fibroin. modified fibroin,
A solvent containing a first solvent as a main component and a second solvent having a boiling point higher than the boiling point of the first solvent as an additive component,
The boiling point of the first solvent is 40°C or more and 120°C or less, and the boiling point of the second solvent is 80°C or more and 280°C or less,
A fibroin solution , wherein the mass ratio of the content of the first solvent to the content of the second solvent is 95/5 or more . The fibroin solution according to claim 7 , wherein the modified fibroin is a modified fibroin derived from an insect or arachnid. The fibroin solution according to claim 7 or 8 , which is used for dry spinning.

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