JP2020122249A - Method for producing fibroin fiber and fibroin solution - Google Patents

Abstract

To obtain a fibroin fiber having excellent tensile strength.SOLUTION: A method for producing a fibroin fiber includes the steps of spinning, by dry spinning, a dope liquid that contains fibroin, and a solvent containing a first solvent as the main component and containing a second solvent having a higher boiling point than that of the first solvent as an additional component.SELECTED DRAWING: None

Description

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

In various industrial fields, fibroin fiber is drawing attention as a new material with high utility value. Regenerated silk fibroin fibers and spider silk fibroin fibers have been conventionally known as fibroin fibers. As a method for producing these fibroin fibers, a large number of wet spinning methods and dry wet spinning methods have been reported. However, in those manufacturing methods, it is necessary to use a large amount of liquid in the coagulation bath and the washing bath, and there is a limit to the improvement in productivity.

As the dry spinning method, for example, a method in which a dope prepared by dissolving silkworm silk fibroin after refining in a mixed organic solvent of calcium chloride and formic acid is discharged into the air from a syringe to form fibers (Non-Patent Document 1), A method (non-patent document 2) of forming fibers by discharging a dope prepared by dissolving silkworm silk fibroin after refining in hexafluoroisopropanol (HFIP) into the air from a syringe has been reported.

Xiaoxiao Yue et al, Materials Letters, 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 the fibroin fiber obtained by the conventional dry spinning method.

Then, this invention aims at obtaining the fibroin fiber excellent in 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 that of the first solvent as an additional component is spun by dry spinning. A method for producing fibroin fiber, comprising the step of:
(2) The method for producing fibroin fiber 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 fiber according to (1) or (2), further including the step of drawing the fiber obtained by the above step in water.
(4) The fibroin fiber according to any one of (1) to (3), wherein the boiling point of the first solvent is 40° C. or higher and 120° C. or lower, and the boiling point of the second solvent is 80° C. or higher and 280° C. or lower. Manufacturing method.
(5) The method for producing fibroin fiber according to any of (1) to (4), wherein the fibroin is fibroin derived from insects or arachnids.
(6) The method for producing fibroin fiber according to any one of (1) to (5), wherein the fibroin is a 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 that of the first solvent as an additional 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 boiling point of the first solvent is 40° C. or higher and 120° C. or lower, and the boiling point of the second solvent is 80° C. or higher and 280° C. or lower.
(10) The fibroin solution according to any one of (7) to (9), wherein the fibroin is derived from insects or arachnids.
(11) The fibroin solution according to any one of (7) to (10), wherein the fibroin is a 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, a fibroin fiber having excellent tensile strength can be obtained.

It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. It is a figure which shows distribution of the value of z/w (%) of fibroin derived from nature. It is a figure which shows the distribution of the value of x/y (%) of fibroin derived from nature. It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. It is a schematic diagram which shows an example of the domain sequence of a modified fibroin.

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

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

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

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

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, 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, 280° C. or lower, 260° C. or lower, 240° C. Hereinafter, it may be 230°C or lower, 220°C or lower, 210°C or lower, or 200°C or lower. As the second solvent, N,N-dimethylformamide, formic acid, dimethylsulfoxide, N,N-dimethylacetamide, propylene carbonate, imidazole, 1-methylimidazole, ionic liquid (1-butyl-3-methylimidazolium chloride or 1-ethyl-3-methylimidazolium diethyl phosphate etc.) and the like.

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 preferably dissolved in the solvent to obtain tensile strength. From the viewpoint of obtaining an excellent fibroin fiber, for example, it may be 95/5 or more, 96/4 or more, 97/3 or more, and 98/2 or more. The mass ratio may be 99/1 or less.

Since the fibroin solution in the present embodiment contains the first solvent having a low boiling point and the second solvent having a high boiling point, the fiber having excellent tensile strength can be obtained by performing dry spinning using this fibroin solution. Raw material fiber) is obtained. Moreover, since this raw material fiber can be drawn in water, the strength thereof can be further improved. The reason for this is not clear, but the present inventors presume as follows.
That is, in order to improve the tensile strength by the secondary stretching, it is necessary to plastically deform the fibroin molecule to increase the rearrangement (orientation) of the molecular chain in the fiber axis. Since water serves as a plasticizer for fibroin, it can be expected to have the effect of promoting molecular orientation during stretching but not excessively promoting crystallization. When the regenerated fibroin fiber has a low crystallinity during drawing in water, the amount of water that serves as a plasticizer is too large and is easily broken (fused) due to swelling. Conversely, when the crystallinity is too high, Due to the strong cohesive force of fibroin, plastic deformation is hindered and rearrangement of molecular chains becomes difficult. In dry spinning using only the first solvent, only regenerated fibers having low crystallinity can be obtained, so that the fibers are melt-fused in water. On the other hand, when the second solvent having a boiling point higher than that of the first solvent is contained, the second solvent remaining in the fiber serves as a plasticizer to bring the mobility of the fibroin molecule, thereby promoting the crystallinity, Secondary stretching is possible without melting even in water. Therefore, by using the second solvent in addition to the first solvent, a fibroin fiber having appropriate crystallinity and excellent tensile strength can be produced, and in-water drawing becomes possible, and fibroin having higher strength can be produced. Fibers can be obtained.

The fibroin may be, for example, one or more 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 the silk fibroin and the spider silk fibroin are used in combination, the ratio of the 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 the spider silk fibroin.

Silk thread is a fiber (cocoon thread) obtained from a cocoon made by a silkworm, Bombyx mori larva. Generally, one cocoon thread is composed of two silk fibroins and a glue (sericin) that covers them from the outside. Silk fibroin is composed of numerous fibrils. Silk fibroin is covered with four layers of sericin. Practically, a silk filament obtained by dissolving and removing the outer sericin by refining is used for clothing. A general silk thread has a specific gravity of 1.33, a fineness of 3.3 decitex on average, and a fiber length of about 1300 to 1500 m. Silk fibroin can be obtained by using natural or silkworm cocoons or used or discarded silk cloth as a raw material.

The silk fibroin may be sericin-removed silk fibroin, sericin-unremoved silk fibroin, or a combination thereof. The sericin-removed silk fibroin is purified by removing sericin covering silk fibroin and other fats. The silk fibroin purified in this way is preferably used as a lyophilized powder. The sericin-unremoved silk fibroin is unpurified silk fibroin in which sericin and the like have not been removed.

The 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 vesicular guideline thread protein, weft thread protein, and vesicular gland protein. Since the large yarn ejection guide thread has a repeating region including a crystalline region and an amorphous region (also referred to as an amorphous region), it has high stress and elasticity. The weft thread of the spider thread is characterized by not having a crystalline region but having a repeating region composed of an amorphous region. The weft thread is less in stress than the large spit tube bookmark thread, but has high elasticity.

The large vesicle dragline protein is produced in the large bottle-shaped line of a spider, and has a characteristic of excellent toughness. Examples of the large vesicle dragline silk proteins include large ampullate spidroins MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Aranius diadematus. ADF3 is one of the two major bookmark thread proteins of the Japanese spider. The polypeptides derived from natural spider silk proteins may be polypeptides derived from these bookmark silk proteins. The polypeptide derived from ADF3 is relatively easy to synthesize and has excellent properties in terms of strength and elongation and toughness.

Weft thread proteins are produced in the flagelliform gland of the spider. Examples of the weft thread protein include flagelliform silk protein derived from Nephila clavipes.

The fibroin may be modified fibroin (recombinant fibroin). The modified fibroin is a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m -(A) _n motif. The modified fibroin may have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.

In the present specification, the “modified fibroin” may be a fibroin whose domain sequence is different from the amino acid sequence of naturally-occurring fibroin or may be the same fibroin as the amino acid sequence of naturally-occurring fibroin. .. The “naturally-derived fibroin” as used herein is also represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a domain sequence.

The “modified fibroin” may be one that uses the amino acid sequence of naturally-derived fibroin as it is, or one that has its amino acid sequence modified depending on the amino acid sequence of naturally-derived fibroin (for example, a cloned naturally-derived fibroin). The amino acid sequence may be modified by modifying the gene sequence of fibroin), or artificially designed and synthesized regardless of naturally-occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence It may have a desired amino acid sequence by chemical synthesis).

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

Modified fibroin is, for example, a modification of the amino acid sequence corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues with respect to the cloned gene sequence of naturally-occurring fibroin. Can be obtained at Amino acid residue substitutions, deletions, insertions and/or additions can be performed by methods well known to those skilled in the art such as partial 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. Yes, specifically, for example, fibroin produced by insects or arachnids can be mentioned.

Examples of fibroin produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamai cya cya pyramid, Anteraea pyramyi, Ori a pu rye, Pomegranate (Anteraea periyi), Pomegranate (Anteraea periyi), Anteraea peryany (Etera peri ny) ), a silk protein produced by silkworms such as Anthera ea limata (Antheraea assama), and a silk protein produced by silkworms such as Anthera ea limata (Antheraea assama). Hornet silk protein is mentioned.

More specific examples of fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence) and AAA278840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include spiders belonging to the genus Araneus (genus Araneus) such as Onigumo, Niwaonigamo, Akanonigumo, Aonigumo and Maeoniguimo, spiders such as Yamashiroonigomo, Jononigu spp. Spiders belonging to the genus Proton, spiders belonging to the genus Pronus, spiders belonging to the genus Cyrtarachne (genus Cyrtarachne), such as spider spider, spider spider Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius such as Mamei Taiseki spider and Mutsugai spider, and belonging to the genus Argiopsis such as Argiogiope, Argiope spp. , Spiders belonging to the genus Cytophora, such as Spiders belonging to the genus Acusilas, such as Spiders belonging to the genus Arachnura, Spiders belonging to the genus Cytophora such as Spiders, Spiders, and Black Spiders. Spider silk protein produced by spiders belonging to the genus Cyclosa (genus Cyclosa), such as spiders belonging to the category ), spider silk proteins, and spider silk spiders belonging to the genus Chorizopes, such as the spider silk spider. Spiders belonging to the genus Tetragnatha, such as the herring-tailed spider, sword-tailed duck spider, and urocore, and the spider belonging to the genus Tetragnatha, the genus Leucaug, which belongs to the genus Leucaug, such as the genus Leucagu Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira, such as black spiders, spiders belonging to the genus Dyschiriognatha, such as dwarf spiders, such as the black-breasted spider, black widow spider, and the spider, Latrodectus mactans. Spiders produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus (genus Latrorectus) and spiders belonging to the genus Euprosthenops (genus Euprosthenops). Examples include pider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

As a more specific example of the spider silk protein produced by spiders, for example, fibroin-3 (adf-3) [from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes 50 (session amino acid sequence No. AGen04)] ), U37520 (base sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein Nevalina spirain spiroline 2 (base sequence). No. AAL32472 (amino acid sequence), AF441245 (base sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ9733155 (base sequence)), and major 2 amperoid ampullate ampulate. (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession No. AAC14589. 1 (amino acid sequence)), minor ampulate NPurolate. clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nephilengys cruentata] (GenBank Accession). No. ABR37278.1 (amino acid sequence) and the like.

More specific examples of naturally-derived fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, among sequences containing INV as DIVISION among the sequence information registered in NCBI GenBank, spidroin, complete, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific product from CDS, and a sequence in which a specific character string is described from SOURCE to TISSUE TYPE.

The modified fibroin may be modified silk (silk) fibroin (a modified silkworm-produced silk protein amino acid sequence), modified spider silk fibroin (spider-produced spider silk protein amino acid sequence is modified) Thing). As the modified fibroin, modified spider silk fibroin is preferable.

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

Examples of the first modified fibroin include a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, the number of amino acid residues of (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, and an integer of 10 to 20. Is even more preferable, an integer of 4 to 16 is even more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the first modified fibroin, in Formula 1, the number of amino acid residues constituting REP is preferably 10 to 200 residues, more preferably 10 to 150 residues, and more preferably 20 to 100 residues. Is more preferable, and 20 to 75 residues are even more preferable. The first modified fibroin has a total number of amino acid residues of glycine residues, serine residues, and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m. The total number is preferably 40% or more, more preferably 60% or more, still more preferably 70% or more.

The first modified fibroin comprises a unit of the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m , and the C-terminal sequence is the amino acid sequence represented by any of SEQ ID NOs: 1 to 3, or It may be a polypeptide which is 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 amino acids of the C-terminal 50 residues of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID NO: 3 has 29 residues removed from the C-terminus with the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.

As a more specific example of the first modified fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 and 90 Mention may be made of modified fibroin which comprises an amino acid sequence having a sequence identity of greater than or equal to %. The sequence identity is preferably 95% or more.

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

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

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

The second modified fibroin has a domain sequence that is different from naturally-occurring fibroin in that 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). At least one motif sequence selected from the group consisting of (1) and (2) 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. May be.

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

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

The second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is more preferable, 4% or less is still more preferable, and 2% or less is particularly preferable. 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 of calculating the content ratio (z/w) of the amino acid sequence consisting of XGX below.

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

Here, z/w in naturally occurring fibroin will be described. First, as described above, when confirmed by a method exemplifying fibroin having amino acid sequence information registered in NCBI GenBank, 663 kinds of fibroins (of which 415 kinds of fibroins derived from arachnids) were extracted. Of all the extracted fibroins, naturally-occurring origin, which contains the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and the content of the amino acid sequence consisting of GGX in the fibroin is 6% or less. Z/w was calculated from the amino acid sequence of fibroin of No. 1 by the above-mentioned calculation method. The result is shown in FIG. The horizontal axis of FIG. 2 represents z/w (%), and the vertical axis represents frequency. As is clear from FIG. 2, the z/w of naturally-derived fibroin is less than 50.9% (50.86% at the highest).

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

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

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

As a more specific example of the second modified fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met). -PRT799) or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.

The modified fibroin of (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 6 is obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin with GQX. The amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 6 in which every two (A) _n motifs are deleted from the N-terminal side toward the C-terminal side, and further before the C-terminal sequence. One [(A) _n motif-REP] was inserted into. The amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) _n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a part of glutamine (Q) residue. The amino acid was substituted with a serine (S) residue and a part of amino acids on the C-terminal side was deleted so that the molecular weight was almost the same as that of SEQ ID NO:7. The amino acid sequence represented by SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a predetermined hinge sequence and a His tag sequence are added to the C-terminal of the sequence repeated 4 times.

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

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

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

The second modified fibroin may include a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection and visualization of the modified fibroin.

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

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

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

Further, a tag sequence that can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence is cleaved can be recovered.

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

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

The modified fibroin of (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 of (2-iv) contains an amino acid sequence having 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. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

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

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

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

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

The third modified fibroin has a domain sequence of 1 to 3 (A) _n motifs at least from the N-terminal side to the C-terminal side as compared with naturally-occurring fibroin (A) _n motifs. May have an amino acid sequence corresponding to the deletion.

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

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

The third modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and has two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -REP] units are sequentially compared in number of amino acid residues, and when the number of amino acid residues in REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to When the maximum value of the sum total of the number of amino acid residues of two adjacent [(A) _n motif-REP] units that are 11.3 is x and the total number of amino acid residues of the domain sequence is y In addition, x/y may have an amino acid sequence of 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. It is more preferable that it is 100%, and it is even more preferable that it is 100% (meaning that it is composed of only alanine residues).

The method of calculating x/y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from modified fibroin. The domain sequence is (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n from the N-terminal side (left side). Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) _{It has an n} motif sequence.

Two adjacent [(A) _n motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, an unselected [(A) _n motif-REP] unit may be present. In FIG. 1, 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 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP, and 4th REP and 5th REP), pattern 4 (1st REP) (Comparison of the second REP). There are other selection methods besides this.

Next, for each pattern, the number of amino acid residues of each REP in two adjacent [(A) _n motif-REP] units selected is compared. The comparison is performed by determining the ratio of the number of other amino acid residues when the number of amino acid residues having a smaller number is 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50=2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is 1, the fifth REP The ratio of the number of amino acid residues is 30/20=1.5.

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

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

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

In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, even more preferably 70% or more. It is even more preferably 75% or more, still more preferably 80% or more. The upper limit of x/y is not particularly limited, and may be 100% or less, for example. When the notch ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the notch ratio is 1:1.8 to 3.4, x/y is x. /Y is preferably 77.1% or more, and when the notch ratio is 1:1.9 to 8.4, x/y is preferably not less than 75.9% and the notch ratio is 1 In the case of 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 a plurality in the domain sequence are composed of only alanine residues, x/y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is still more preferable, 60% or more is still more preferable, 70% or more is still more preferable, 80% or more is more preferable. It is particularly preferable that The upper limit of x/y is not particularly limited and may be 100% or less.

Here, x/y in naturally occurring fibroin will be described. First, as described above, when confirmed by a method exemplifying fibroin having amino acid sequence information registered in NCBI GenBank, 663 kinds of fibroins (of which 415 kinds of spider-derived fibroins) were extracted. Among all the extracted fibroins, x/y was calculated from the amino acid sequence of naturally-derived fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _m by the above-mentioned calculation method. Was calculated. The results when the serrated ratio is 1:1.9 to 4.1 are shown in FIG.

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

The third modified fibroin has, for example, one or more of the sequences encoding the (A) _n motif deleted from the cloned gene sequence of naturally-derived fibroin so that x/y is 64.2% or more. It can be obtained. In addition, for example, an amino acid sequence corresponding to the deletion of one or more (A) _n motifs is designed and designed from the amino acid sequence of naturally occurring fibroin so that x/y is 64.2% or more. It can also be obtained by chemically synthesizing a nucleic acid encoding the above amino acid sequence. In any case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and/or added. The amino acid sequence corresponding to the above may be modified.

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

The x/y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-derived fibroin) at the Giza ratio of 1:1.8 to 11.3 is 15.0%. The values of x/y in the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%. The value of x/y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%. The values of z/w in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.

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

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

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

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

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

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

The modified fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by the 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 from the N-terminal side to the C-terminal side. Toward the direction, the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is 1, The maximum value of the sum total of the number of amino acid residues of two adjacent [(A) _n motif-REP] units whose ratio of the number of amino acid residues of REP is 1.8 to 11.3 is x. When the total number of amino acid residues in the domain sequence is y, x/y is preferably 64.2% or more.

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

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

As a more specific example of the fourth modified fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410). ), 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. A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by Specific embodiments of the modified fibroin containing the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

The fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a large hydrophobicity index, and/or REP as compared with naturally-derived fibroin. It may have an amino acid sequence locally containing a region with a large hydrophobicity index, which corresponds to the insertion of one or more amino acid residues with a large hydrophobicity index.

It is preferable that the region having a locally large hydrophobicity index is composed of consecutive 2 to 4 amino acid residues.

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

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

The fifth modified fibroin has, for example, one or more hydrophilic amino acid residues (for example, an amino acid residue having a negative hydrophobicity index) in REP from the gene sequence of the cloned naturally-derived fibroin, and a hydrophobic amino acid residue. It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid residues in REP. Further, 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 occurring fibroin, and/or one or more hydrophobic amino acid residues in REP. It can also be obtained by designing an amino acid sequence corresponding to the insertion of the nucleotide sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally-derived fibroin, and/or one or more hydrophobic amino acids in REP. In addition to the modification corresponding to the insertion of a residue, the modification of the amino acid sequence corresponding to the replacement, deletion, insertion and/or addition of one or more amino acid residues may be performed.

The fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and extends from the (A) _n motif located closest to the C terminus to the C terminus of the above domain sequence. In all REPs contained in the sequence excluding the sequence from the domain sequence, the total number of amino acid residues contained in the region where the average hydrophobic index of 4 consecutive amino acid residues is 2.6 or more is p, When the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located closest to the C terminus to the C terminus of the domain sequence is q, the p/q is 6 It may have an amino acid sequence of 2% or more.

Regarding the hydrophobic index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic charactor of a pro. 105-132). Specifically, the hydrophobicity index (hydropathic 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 described in more detail. For the calculation, a sequence obtained by removing the sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m to the C-terminal of the domain sequence (A) _n motif located at the most C-terminal side. (Hereinafter, referred to as “array A”) is used. First, in all REPs contained in the 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 obtained by dividing the total HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to multiple “4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more”, it must be included as 1 amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in Sequence A is q.

For example, when 20 consecutive “4 consecutive amino acid residues having an average hydrophobicity index of 2.6 or more” are extracted (no overlap), the average hydrophobicity index of 4 consecutive amino acid residues is 2 A region of 0.6 or more includes 20 consecutive 4 amino acid residues (no duplication), and p is 20×4=80. Further, for example, when two “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” overlap by 1 amino acid residue, the hydrophobicity index of the consecutive 4 amino acid residues The region having an average value of 2.6 or more contains 7 amino acid residues (p=2×4-1=7. “−1” is a deduction for the overlap). For example, in the case of the domain sequence shown in FIG. 4, since seven “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” do not overlap, p is 7×4= 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 ((A) _n motif present at the end on the C-terminal side is not included). Next, p/q(%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170=16.47%.

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

The fifth modified fibroin includes, for example, one or more hydrophilic amino acid residues (for example, a hydrophobicity index) in REP such that the amino acid sequence of cloned naturally-occurring fibroin satisfies the above p/q condition. Is replaced with a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index), and/or one or more hydrophobic amino acid residues are inserted into REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Alternatively, for example, it can be obtained by designing an amino acid sequence satisfying the above p/q condition from the amino acid sequence of naturally-derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more amino acid residues in REP were replaced with amino acid residues having a large hydrophobicity index, and/or one or more amino acid residues in REP were compared to naturally-occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, a modification corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues may be carried out. ..

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

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

The modified fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 19 is composed of 3 amino acid residues in every other REP except for the C-terminal terminal domain sequence with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410). An amino acid sequence (VLI) was inserted at two positions, a part of glutamine (Q) residue was replaced with a serine (S) residue, and a part of C-terminal side amino acid was deleted. The amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with an amino acid sequence (VLI) consisting of 3 amino acid residues at every other REP inserted at one position. is there. The amino acid sequence represented by SEQ ID NO: 21 is the amino acid sequence represented by SEQ ID NO: 8 with two amino acid sequences (VLI) each consisting of three amino acid residues inserted every other REP.

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

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

The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located at the most C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C terminus of the domain sequence from the domain sequence 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 located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, , P/q is preferably 6.2% or more.

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

As a more specific example of the modified fibroin containing a tag sequence, (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv ) A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 can be mentioned.

The amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including sequences and hinge sequences).

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

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

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located at the most C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C terminus of the domain sequence from the domain sequence 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 located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, , P/q is preferably 6.2% or more.

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

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

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

When the sixth modified fibroin contains a GPGXX motif in the 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 the present specification, the “GPGXX motif content rate” 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 comprising a domain sequence represented by _n motifs (modified fibroin or natural origin (A) _n motif located at the most C-terminal side in the fibroin) to the C-terminal of the domain sequence in all REPs contained in the sequence, the number of GPGXX motifs contained in that region Let s be the number three times the total number (that is, the total number of G and P in the GPGXX motif), and the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. And the total number of amino acid residues of all REPs excluding (A) _n motifs is t, the GPGXX motif content is calculated as s/t.

In the calculation of the GPGXX motif content, "the sequence in which the sequence from the (A) _n motif located at the most C-terminal to the C-terminal of the domain sequence is removed from the domain sequence" is "the most C-terminal side". The sequence (A) from the _n- motif to the C-terminal of the domain sequence" (sequence corresponding to REP) may include a sequence having low correlation with the characteristic sequence of fibroin, and m is small. In this case (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content rate, and is for eliminating this effect. When the “GPGXX motif” is located at the C-terminal of REP, it is treated as a “GPGXX motif” even if “XX” is “AA”.

FIG. 5 is a schematic diagram showing a domain sequence of modified fibroin. A method of calculating the GPGXX motif content rate will be specifically described with reference to FIG. First, in the modified fibroin domain sequence (“[(A) _n motif-REP] _m- (A) _n motif” type.) shown in FIG. 5, all REPs are “most C-terminally located. (A) The sequence from the _n- motif to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence shown as “Region A” in FIG. 5). The number of GPGXX motifs is 7, and s is 7×3=21. Similarly, all REPs are "sequences obtained by excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (sequence shown as "region A" in FIG. 5). .), the total number t of amino acid residues of all REPs excluding the (A) _n motif from the sequence is 50+40+10+20+30=150. Then, s/t (%) can be calculated by dividing s by t, which is 21/150=14.0% in the case of the modified fibroin of FIG.

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

In the present specification, the "glutamine residue content rate" 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 comprising a domain sequence represented by _n motifs (modified fibroin or natural origin In (fibroin), all contained in the sequence (sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the total number of glutamine residues contained in the region is defined as u, the sequence from the (A) _n motif located closest to the C terminus to the C terminus of the domain sequence is removed from the domain sequence, and (A) _n When the total number of amino acid residues of all REPs excluding the motif is t, the glutamine residue content rate is calculated as u/t. In the calculation of the glutamine residue content rate, the reason why “the sequence in which the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence” is the target is the same as the above-mentioned reason. It is the same.

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

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

As shown in Table 1, as amino acid residues having a larger hydrophobicity index than glutamine residues, isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) An amino acid residue selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H) can be mentioned. it can. Among these, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. More preferably, it is an amino acid residue selected from 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, further preferably 0 or more, and 0.3 or more. Is more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present 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 comprising a domain sequence represented by _n motifs (modified fibroin or natural origin In (fibroin), all contained in the sequence (sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP of, 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 closest to the C-terminal to the C-terminal 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 of all REPs excluding _n motifs. In the calculation of the hydrophobicity of REP, the reason why "the sequence in which the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence" is the target is the same as the above-mentioned reason. It is the same.

The sixth modified fibroin has a domain sequence in which one or more glutamine residues in REP are deleted, and/or one or more glutamine residues in REP, as compared to naturally-occurring fibroin. In addition to the modification corresponding to substituting with another amino acid residue, there may be modification of the amino acid sequence corresponding to substituting, deleting, inserting and/or adding one or more amino acid residues. ..

The sixth modified fibroin includes, for example, deleting one or more glutamine residues in REP from the cloned gene sequence of naturally-derived fibroin, and/or deleting one or more glutamine residues in REP from each other. It can be obtained by substituting the amino acid residue of Further, for example, one or more glutamine residues in REP have been deleted from the amino acid sequence of naturally-occurring 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 the corresponding amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As a more specific example of the sixth modified fibroin, (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met). -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698) or SEQ ID NO: 32 (Met-PRT966), or a modified fibroin. (6-ii) 90% or more sequence identity with 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. The modified fibroin containing the amino acid sequence which it has can be mentioned.

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

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

The amino acid sequence represented by SEQ ID NO: 32 is a sequence obtained by repeating twice the region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410), and substituting VF for VF, The remaining Q is replaced with I.

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

The modified fibroin of (6-i) consists of 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 may be.

The modified fibroin of (6-ii) has 90% or more amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. It includes an amino acid sequence having the sequence identity of. The modified fibroin of (6-ii) also has a domain represented by the formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m -(A) _n motif. A protein that contains a sequence. The sequence identity is preferably 95% or more.

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

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

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

The amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 are SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, respectively. , SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) represented by SEQ ID NO: 11 was added to the N-terminal of the amino acid sequence represented by SEQ ID NO: 32. It is a thing. Since only the tag sequence was added to the N-terminal, the glutamine residue content did not change, 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. And the amino acid sequence represented by SEQ ID NO: 40 all have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) consists of the amino acid sequence 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 or SEQ ID NO:40. It may be.

The modified fibroin of (6-iv) has 90% or more amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. It includes an amino acid sequence having the sequence identity of. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. A protein that contains a sequence. The sequence identity is preferably 95% or more.

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

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

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

<Method for producing modified fibroin>
The modified fibroin according to any of the above embodiments, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by utilizing a gene encoding natural fibroin, amplifying it by polymerase chain reaction (PCR) or the like, cloning it, and modifying it by a genetic engineering method, or chemically synthesizing it. can do. The method of chemically synthesizing nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of fibroin obtained from the NCBI web database or the like, AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) or the like can be used. A gene can be chemically synthesized by a method of ligating automatically synthesized oligonucleotides by PCR 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 obtained by adding an amino acid sequence consisting of a start codon and a His10 tag to the N-terminal of the above amino acid sequence is synthesized. You may.

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

The type of expression vector can be appropriately selected according to 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 can autonomously replicate in the host cell or can be integrated into the host chromosome 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 prokaryote and eukaryote such as yeast, filamentous fungus, insect cell, animal cell and plant cell can be preferably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. Examples of the microorganism belonging to the genus Escherichia include Escherichia coli. Examples of the microorganism 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 ammoniaphilum and the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes and the like. Examples of the microorganism belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, examples of a vector for introducing a nucleic acid encoding modified fibroin include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238569) and the like can be mentioned.

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

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

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

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

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

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

Cultivation of prokaryotic organisms such as Escherichia coli or eukaryotic organisms such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration agitation culture. The culture temperature is, for example, 15 to 40°C. Cultivation time is usually 16 hours to 7 days. The pH of the culture medium during culturing 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 and the like.

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

The expressed modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed intracellularly in a lysed state, after culturing, the host cells are recovered by centrifugation, suspended in an aqueous buffer solution, and then ultrasonically disrupted, French press, Menton. A cell-free extract is obtained by crushing host cells with a Gaurin homogenizer, Dynomill and the like. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolation and purification of proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent is used. Precipitation method, anion-exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), and cation using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography, hydrophobic chromatography using butyl sepharose, phenyl sepharose, etc. resins, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoretic methods such as isoelectric focusing The above method can be used alone or in combination to obtain a purified preparation.

When the modified fibroin is expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to recover the insoluble substance of the modified fibroin as a precipitate fraction. The recovered insoluble body of modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified fibroin preparation can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a purified preparation can be obtained by treating the culture with a technique such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

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 with respect to 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 the fibroin solution is used as the spinning solution, the viscosity is not particularly limited as long as the viscosity allows dry spinning, but from the viewpoint of productivity, the viscosity of the fibroin solution at 25° C. is, for example, 10,000 mPa·sec. 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, It may be 000 mPa·sec or less, or 15,000 mPa·sec or less. The viscosity of the fibroin solution can be measured using, for example, a product name “EMS viscometer” manufactured by Kyoto Electronics Manufacturing Co., Ltd.

The fibroin solution may further contain various additives, if necessary. Examples of the additive include a plasticizer, a leveling agent, a cross-linking agent, a crystal nucleating agent, an antioxidant, an ultraviolet absorber, a colorant, a filler, and a synthetic resin. 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 fibroin-containing fibers (fibroin fibers), and is used for spinning (particularly preferably dry spinning) fibers containing fibroin (fibroin fibers). It is particularly suitable as a solution (dope solution or spinning solution).

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

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

The method for discharging the fibroin solution (spinning solution) from the spinneret is not particularly limited, but, for example, a method using a metering pump can be used as a means for sending the fibroin solution (spinning solution). The discharge amount can be appropriately adjusted according to 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 appropriately adjusted depending on the concentration and viscosity of the fibroin solution used, the type of solvent, and the like. The temperature is preferably 30°C to 100°C from the viewpoint of preventing deterioration of fibroin and the like. The temperature is higher than the boiling point of the solvent in the fibroin solution from the viewpoint of reducing the pressure increase due to volatilization of the solvent and the possibility of clogging in the pipe due to solidification of the fibroin solution, and the melting point of the solvent. It is preferable to set the above temperature to the lower limit. This improves the process stability.

The spinneret shape, hole shape, number of holes, etc. are not particularly limited, and can be appropriately selected according to the desired fiber diameter, the number of single yarns, and the like. For example, when the hole shape of the spinneret is circular, a hole diameter of 0.01 mm or more and 0.6 mm or less can be exemplified. When the hole diameter is 0.01 mm or more, pressure loss can be reduced and equipment cost can be suppressed. When the pore diameter is 0.6 mm or less, it is possible to reduce the need for a stretching operation for thinning the fiber diameter, and reduce the possibility of stretching breakage between the discharge and the take-up.

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

The fibroin fiber obtained by the spinning step may be used as it is for any purpose. Alternatively, a step of drawing the fibroin fiber (drawing step) may be further performed to obtain a fibroin fiber drawn at an arbitrary draw ratio, and the obtained fibroin fiber may be used for any purpose.

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

The drawing method is preferably wet heat drawing, and more preferably underwater drawing in which the fibroin fiber is drawn in water, from the viewpoint that a fibroin fiber more excellent in strength can be obtained. 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. ..

The dry heat drawing can be performed by using a device such as a contact type hot plate and a non-contact type furnace, but is not particularly limited, and the fiber is heated to a predetermined temperature and a predetermined temperature is set. Any device capable of stretching at a 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 in °C.

The draw ratio in the drawing step may be, for example, 1 to 30 times, 1 to 25 times, or 1 to 20 times that of the raw material fibers (undrawn yarn or predrawn yarn). , 1 to 15 times, 1 to 10 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, 2 to 4 times And may be 2-3 times.

In the stretching step, wet heat stretching (underwater stretching) and dry heat stretching may be performed individually, or may be performed in multiple stages or in combination. That is, as a stretching step, 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 the third stage is further performed. The wet heat stretching and the dry heat stretching can be appropriately combined such that the stretching is performed by the dry heat stretching.

The lower limit value of the final draw ratio of the fibroin fiber that has undergone the drawing process is, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times that of the raw material fiber (undrawn yarn or pre-drawn yarn). It may be any of double, 7×, 8×, or 9×. The upper limit of the final draw ratio of the spider silk fibroin fiber that has undergone the drawing 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 draw ratio may be 3 to 40 times, 3 to 30 times, 5 to 30 times, 5 to 20 times, 5 to 15 times. And may be 5 to 13 times. However, the draw ratio is not limited as long as the desired fiber thickness, mechanical properties and other characteristics can be obtained.

Before or after the stretching step, an oil agent may be added to the fiber for the purpose of imparting antistatic property, sizing property, lubricity and the like, if necessary. The type and amount of the oil agent to be applied are not particularly limited, and can be appropriately adjusted in consideration of the use of the fiber, handleability of the fiber, and the like.

[Product]
The product according to the present embodiment may be various products containing the above-mentioned fibroin fiber. Products can include, for example, fibers, threads, fabrics, knits, braids, nonwovens, paper, and cotton. Examples of the fiber include long fiber, short fiber, monofilament, multifilament, and the like. Examples of the yarn include spun yarn, twisted yarn, false twisted yarn, processed yarn, mixed yarn, mixed yarn, and the like. You can Further, from these fibers and yarns, it is possible to manufacture fabrics such as woven fabrics, knitted fabrics, braids, non-woven fabrics, paper, cotton and the like. These products can be manufactured by a known method.

Hereinafter, the present invention will be described 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 (GenBank Accession Nos.: P46804.1, GI:1174415) derived from Nephila clavipes, a modified spider silk fibroin (SEQ ID NO: 40) Hereinafter, also referred to as "PRT966") was designed. The amino acid sequence represented by SEQ ID NO: 40 is different from the amino acid sequence derived from Nephila clavipes, while the amino acid sequence represented by SEQ ID NO: 40 is the amino acid represented by SEQ ID NO: 7 for the purpose of improving hydrophobicity. In the sequence, the region of 20 domain sequences existing in the sequence is repeated twice, and the sequence has a sequence in which QQ is all replaced with VF and the remaining Q is replaced with I. The amino acid sequences shown (tag sequence and hinge sequence) are added.

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

(2) Expression of modified fibroin Escherichia coli BLR(DE3) was transformed with the expression vector obtained in (1). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that the OD ₆₀₀ was 0.005. The temperature of the culture solution was maintained 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 solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. The dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturated concentration, and the culture was carried out for 20 hours. Then, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce the expression of modified fibroin. At 20 hours after the addition of IPTG, the culture solution was centrifuged to collect the bacterial cells. SDS-PAGE is performed using the cells prepared from the culture medium before and after the addition of IPTG, and the appearance of the target artificial structural protein due to the appearance of the modified fibroin-sized band of interest depending on the addition of IPTG. confirmed.

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

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

<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.

[Production of fibroin fiber]
12. The fibroin solutions of Examples 1-1 to 1-3, Reference Examples 1-1 to 1-3 and Comparative Example 1 were attached to the tip of the spinning syringe filled with the solutions, Φ0.25 mm, length: 12. It was discharged through a 7 mm or 25.4 mm nozzle. The formed yarn is wound at a speed of 1.0 m/min or 2.5 m/min on a first roller arranged 50 cm below the nozzle, and wound on a winder at the same speed via a second roller. I took it. The fibroin fiber thus produced was stored in a constant temperature and constant humidity environment (temperature 20±2° C., relative humidity 65±2%).

[Evaluation of fibroin fiber]
The tensile strength of the obtained fibroin fiber was measured using a tensile tester (INSTRON 3342) under the condition of relative humidity of 65%. The tensile strength was evaluated by a relative value when the tensile strength when only HFIP was used as the solvent (Comparative Example 1) was 100. The relative values are shown in Table 6.

Further, the obtained fiber was placed on the same day or in an environment of 20° C. and 65% RH for 1 day, and then stretched in water. In the underwater drawing, a continuous drawing machine equipped with a feed roller and a take-up roller is used, the temperature of water during drawing is 20° C., the length of the fiber immersed in water is 30 cm, and the draw ratio is 1 to 3 times. And the conditions. Immediately after stretching, the film was wound on a hot roller at 60° C. and dried, and then wound by a winder. The suitability for underwater stretching was evaluated according to the following criteria. The results are shown in Table 6.
A: Underwater drawing was possible and the drawn fiber could be collected for 5 minutes or longer.
B: Underwater drawing was possible and the drawn fiber could be collected for less than 5 minutes.
C: Underwater drawing was possible, but fibers after drawing could not be collected.
D: Stretching in water was impossible.

As can be seen from Table 6, in Examples using a solvent in which HFIP is mixed with a solvent having a higher boiling point than HFIP, a comparative example using only HFIP or a solvent in which HFIP is mixed with a solvent having a lower boiling point than HFIP was used. The tensile strength is significantly improved compared to the reference example. The examples are also excellent in suitability for underwater stretching.

Claims (12)

A step of spinning a dope solution containing fibroin as a main component and a solvent containing a second solvent having a boiling point higher than the boiling point of the first solvent as an additional component by dry spinning A method for producing fibroin fiber, comprising: The method for producing fibroin fiber according to claim 1, wherein a 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 fiber according to claim 1 or 2, further comprising a step of drawing the fiber obtained by the step in water. 4. The fibroin fiber according to claim 1, wherein the boiling point of the first solvent is 40° C. or higher and 120° C. or lower, and the boiling point of the second solvent is 80° C. or higher and 280° C. or lower. Production method. The method for producing fibroin fiber according to any one of claims 1 to 4, wherein the fibroin is fibroin derived from insects or arachnids. The method for producing fibroin fiber according to any one of claims 1 to 5, wherein the fibroin is a modified fibroin. Fibroin,
A fibroin solution containing a solvent containing a first solvent as a main component and a second solvent having a boiling point higher than that of the first solvent as an additional component. The fibroin solution according to claim 7, 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 or 8, wherein the boiling point of the first solvent is 40°C or higher and 120°C or lower, and the boiling point of the second solvent is 80°C or higher and 280°C or lower. The fibroin solution according to any one of claims 7 to 9, wherein the fibroin is derived from insects or arachnids. The fibroin solution according to any one of claims 7 to 10, wherein the fibroin is a modified fibroin. The fibroin solution according to any one of claims 7 to 11, which is used for dry spinning.

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