WO2021065812A1

WO2021065812A1 - Doping liquid and method for producing engineered fibroin molded article using same

Info

Publication number: WO2021065812A1
Application number: PCT/JP2020/036664
Authority: WO
Inventors: オリバーセイエッドシャファート
Original assignee: Ｓｐｉｂｅｒ株式会社
Priority date: 2019-09-30
Filing date: 2020-09-28
Publication date: 2021-04-08

Abstract

The purpose of the present invention is to provide a doping liquid that has excellent solvent resistance, and is useful in production of an engineered fibroin molded article that has a high fineness when the article is in the form of a filament. The doping liquid according to the present invention contains engineered fibroin, formic acid, and at least one transition metal ion selected from the group consisting of an ion, a ruthenium ion, an osmium ion, a cobalt ion, a nickel ion, a copper ion, and a zinc ion.

Description

Doping liquid and method for producing a modified fibroin molded product using the same

The present invention relates to a doping solution and a method for producing a modified fibroin molded product using the same.

Conventionally, silk fibroin fibers, which are recycled silk fibers, modified spider silk fibroin, and the like have been known as fibroin molded bodies. In addition, some doping solutions used in these productions have also been proposed. For example, a dope solution containing a structural protein, a metal compound having a metal atom and an organic group, and a solvent has been proposed (Patent Document 1).

International Publication No. 2019/0660337

In order to develop it into various applications, the dope liquid is required to have excellent solvent resistance in the obtained fibroin molded product. Further, the doping liquid is required to have high fineness of the obtained fibrous fibroin molded product.

An object of the present invention is to provide a doping solution which is excellent in solvent resistance and which is useful for producing a modified fibroin molded product having a high fineness when it is fibrous. Another object of the present invention is to provide a method for producing a fibroin molded product using the above-mentioned doping liquid.

As a result of diligent studies on the above problems, the present inventors have at least selected from the group consisting of modified fibroin and iron ion, ruthenium ion, osmium ion, cobalt ion, nickel ion, copper ion and zinc ion. By using a dope solution containing one kind of transition metal ion and formic acid, it is possible to produce a modified fibroin molded product having excellent solvent resistance and high fineness when it is fibrous. It was discovered for the first time, and the present invention was completed. That is, the present invention relates to, for example, the following inventions.

One aspect of the present invention is a modified fibroin, at least one transition metal ion selected from the group consisting of iron ion, ruthenium ion, osmium ion, cobalt ion, nickel ion, copper ion and zinc ion, and formic acid. With respect to the dope solution, including.

In one embodiment, the modified fibroin may be selected from the group consisting of modified silk fibroin and modified spider silk fibroin.

In one embodiment, the transition metal ion may be selected from the group consisting of cobalt ion, nickel ion and zinc ion.

In one embodiment, the transition metal ion may be a zinc ion.

In one aspect, the modified fibroin may be modified spider silk fibroin.

In one embodiment, the modified fibroin has at least one amino acid residue selected from the group consisting of histidine residue, arginine residue, cysteine residue and serine residue, and the transition metal ion is an amino acid residue. It may be bound to at least a part.

In one embodiment, the modified fibroin has a histidine residue and the transition metal ion may be attached to at least a portion of the histidine residue.

Another aspect of the present invention relates to a method for producing a modified fibroin molded product, which comprises a coagulation step of removing formic acid from the dope solution to obtain a coagulated product of the dope solution.

In one aspect, the coagulation step may be a step of discharging the doping liquid into the coagulation liquid to coagulate it.

In one aspect, the modified fibroin molded article may be a fiber or film containing the modified fibroin.

According to the present invention, it is possible to provide a doping solution which is excellent in solvent resistance and which is useful for producing a modified fibroin molded product having a high fineness when it is in the form of fibers. Further, the present invention can provide a method for producing a modified fibroin molded product using the above-mentioned doping solution.

FIG. 1 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 2 is a diagram showing the distribution of z / w (%) values of naturally occurring fibroin. FIG. 3 is a diagram showing the distribution of x / y (%) values of naturally occurring fibroin. FIG. 4 is a schematic diagram showing an example of the domain sequence of modified fibroin. FIG. 5 is a schematic diagram showing an example of the domain sequence of modified fibroin. It is a graph which shows an example of the result of the moisture absorption heat generation test.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings in some cases. However, the present invention is not limited to the following embodiments.

<Doping liquid>

The dope solution according to the present embodiment includes modified fibroin, at least one transition metal ion selected from the group consisting of iron ion, ruthenium ion, osmium ion, cobalt ion, nickel ion, copper ion and zinc ion, and formic acid. And, including. The doping liquid according to the present embodiment can also be said to be a doping liquid in which modified fibroin and transition metal ions are dissolved in formic acid.

(Modified fibroin)

The modified fibroin according to the present embodiment has 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.} It is a protein contained. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids. In the present embodiment, the modified silk fibroin and the modified spider silk fibroin are preferable, and the modified spider silk fibroin is more preferable, from the viewpoint of excellent heat retention, moisture absorption and heat generation and / or flame retardancy. is there.

As used herein, the term "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be fibroin having the same amino acid sequence as naturally occurring fibroin. “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 the domain sequence to be used.

The "modified fibroin" may be one in which the amino acid sequence of naturally-derived fibroin is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived). It may be an amino acid sequence modified by modifying the gene sequence of fibroin, or an artificially designed and synthesized product that does not depend on 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 fibroin-specific crystalline region (typically _{corresponding to the (A) n} motif of an amino acid sequence) and an amorphous region (typically to the REP of an amino acid sequence). An amino acid sequence that produces (corresponding.)), _{Which is represented by the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, (A) _n motif shows 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, 2 to 27, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (A) _{the ratio of the number of alanine residues to the total number of amino acid residues in the n} motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). A plurality of (A) _n motifs present in the domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160, or It may be an amino acid sequence composed of 1 to 180 amino acid residues. m represents an integer of 2 to 300, 8 to 300 or 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, It may be an integer of 20 to 140 or 20 to 120. 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.

The modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. It can be obtained by modifying. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).

Naturally-derived 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.} Yes, specifically, for example, fibroin produced by insects or arachnids.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pyrai, 蟞蚕 (Anteraea perni), and tussah. ), Silk moth (Samia cinthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama), etc. Hornet silk protein can be mentioned.

More specific examples of insect-produced fibroin include, for example, the silk moth fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).

Examples of fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne), spiders belonging to the genus Trinofundamashi, and spiders belonging to the genus Cyrtarachne. Spiders belonging to (Gasteracantha genus), spiders belonging to the genus Isekigumo (genus Ordgarius) such as Mameitaisekigumo and Mutsutogaysekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc. Spiders belonging to the genus Arachunura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora ) Spiders, spiders, spiders belonging to the genus Cyclosa, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Tetragnatha, such as Yasagata spider, Harabiroashidakagumo, and Urokoa shinagamo, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as spiders, spiders belonging to the genus Dyschiriognatha, spiders such as spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus (Latrodectus) and spiders belonging to the family Spiders (Tetragnathidae) such as spiders belonging to the genus Euprostenops Examples include pider silk protein. Examples of the spider silk protein include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank sequence number AAC4011 (amino acid sequence), U47856 (base sequence)) ), U37520 (base sequence)), major amplifier speedin 1 [derived from Laterectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk proteinaspirin Numbers AAL32472 (amino acid sequence), AF441245 (base sequence)), major protein speedin 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major protein (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor aggregate silk protein 1 [Nephila protein] (GenBank accession number AAC14589.1 (amino acid sequence)), minor amplifier Clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify speedin-like protein [Nefilengys proteina] (GenBank accession number ABR3728.1 (amino acid sequence)) and the like.

As a more specific example of naturally-derived fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidroin, complete, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

The modified fibroin according to the present embodiment may be modified silk fibroin (modified amino acid sequence of silk protein produced by spiders), or modified spider silk fibroin (spider silk protein produced by spiders). It may be a modified amino acid sequence). As the modified fibroin, modified spider silk fibroin (also referred to as “artificial spider silk protein”) is preferable.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large spitting tube bookmarker thread protein produced in the large bottle-shaped gland of spider, and modified fibroin with a reduced content of glycine residues. (Second modified fibroin), (A) _{reduced content of n} motifs Modified fibroin (third modified fibroin), content of glycine residues, and (A) reduced content of _{n motifs} It has a modified fibroin (fourth modified fibroin), a modified fibroin having a domain sequence containing a region having a locally high hydrophobicity index (fifth modified fibroin), and a domain sequence having a reduced content of glutamine residues. Modified fibroin (sixth modified fibroin) can be mentioned.

Examples of the first modified fibroin include proteins 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 2 to 27, further 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, the number of amino acid residues constituting REP in the formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. Is even more preferable, and 20 to 75 residues are even more preferable. In the first modified fibroin, the total number of residues of glycine residue, serine residue and alanine residue contained in the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _{m is the amino acid residue.} It is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number.

The first modified fibroin contains the 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 shown in any of SEQ ID NOs: 1 to 3 or It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

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

As a more specific example of the first modified fibroin, the amino acid sequence shown in (1-i) SEQ ID NO: 4 (recombinant spider silk protein ADF3 KaiLargeNRSH1), or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 and 90 A modified fibroin containing an amino acid sequence having a sequence identity of% or more can be mentioned. The sequence identity is preferably 95% or more.

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

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

The second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues 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 replaced with another amino acid residue as compared with naturally occurring fibroin. ..

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

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

The second modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and is located closest to the C-terminal side of the _{domain sequence (A) from the n} motif to the above domain sequence. The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence. When the total number of amino acid residues in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal 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 with respect to the total number of amino acid residues in the n} motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of 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 even more preferable, 4% or less is even 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 for 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, it is located most on the C-terminal side from the domain sequence (A) _n. The 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 X (center X) contained in two XGX exists as in the case of an amino acid sequence consisting of XGXGX, the 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-terminal side to the C-terminal 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 most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

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

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

The second modified fibroin is, for example, modified from the cloned naturally occurring fibroin gene sequence by substituting at least a part of the base sequence encoding the glycine residue to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be replaced so that z / w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment 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. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be carried out.

The other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than the glycine residue, but is a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and methionine ( Hydrophobic amino acid residues such as M) residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S) ) Residues, hydrophilic amino acid residues such as lysine (K) residue and glutamate (E) residue are preferred, valine (V) residue, phenylalanine (F) residue, leucine (L) residue, isoleucine ( I) Residues and Glutamine (Q) Residues are more preferred, and Glutamine (Q) Residues are 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) -Contains an amino acid sequence represented by PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by (2-ii) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Modified fibroin can be mentioned.

(2-i)
The modified fibroin of is described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. _{In the amino acid sequence shown by SEQ ID NO: 7, every two (A) n} motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence is further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added.} It is substituted with a serine (S) residue and a part of the amino acid on the C-terminal side is deleted. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A hinge sequence and a His tag sequence are added to the C-terminal of the sequence obtained by repeating the above 4 times.

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

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by _{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. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of glycine is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

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

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

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

Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of 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 in which the tag sequence can be separated by 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 has been separated can also be recovered.

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

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

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

The modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 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 the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of glycine is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w 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 according to the type of host.

The third modified fibroin has an amino acid sequence _{whose domain sequence has a reduced content of (A) n motif as compared with 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 more (A) n motifs as compared with the naturally occurring fibroin.}

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

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

_{The third modified fibroin has a domain sequence of at least two consecutive (A) n-} motif deletions and one (A) from the N-terminal side to the C-terminal side as compared to naturally occurring fibroin. ) It may have an amino acid sequence corresponding to the deletion of the _{n-motif being repeated in this order.}

The third modified fibroin may have an amino acid sequence corresponding to the deletion _{of the (A) n} motif at least every other two domain sequences from the N-terminal side to the C-terminal side. ..

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

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n. Motif-Third REP (10 amino acid residues)-(A) _n Motif-Fourth REP (20 amino acid residues)-(A) _n Motif-Fifth REP (30 amino acid residues)-(A) _It has an arrangement called n motifs.

Two adjacent [(A) _n motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, _{there may be a [(A) n} motif-REP] unit that is not selected. In FIG. 1, pattern 1 (comparison between the first REP and the second REP and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. There are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in _{two adjacent [(A) n motif-REP] units selected is compared.} The comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 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 with a smaller number of amino acid residues is set to 1, the fifth REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is 1.8 to 11.3 [(A) _n motif-REP] unit set. Shown by solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other 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, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of _{(A) n motif.} is there.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximized is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

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

In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. 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 7 of the (A) n} motifs present in the domain sequence are composed of only alanine residues, the x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, 70% or more is even more preferable, and 80% or more. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

Here, x / y in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types of arachnid-derived fibroin were extracted) were extracted. Of all the extracted fibroins, x / y from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _{m by the above calculation method.} Was calculated. The results when the jagged ratio is 1: 1.9 to 4.1 are shown in FIG.

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

The third modified fibroin, for example, deletes one or more of the sequences encoding the _{(A) n} motif from the cloned naturally occurring fibroin gene sequence so that x / y is 64.2% or more. Can be obtained by Further, for example, an amino acid sequence corresponding to the deletion _{of one or more (A) n} motifs so that x / y is 64.2% or more is designed and designed from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. _{In each case, in addition to the modification corresponding to the deletion of (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) -Contains an amino acid sequence represented by PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Modified fibroin can be mentioned.

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

The value of x / y in the jagged ratio of 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 15.0%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 17 and the amino acid sequence shown in SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in 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% and 66. 1% and 70.0%.

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

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by _{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 represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _{The amino acid residue of two adjacent [(A) n} motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3) When the maximum value of the total value obtained by adding the radix is x and 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 the tag sequence described above at either or both of the N-terminus and the C-terminus.

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

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

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

The modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 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 the 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 represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _Let x be the maximum value of the total value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues in REP is 1.8 to 11.3. , 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 according to the type of host.

The fourth modified fibroin has an amino acid sequence whose domain sequence has a _{reduced content of (A) n} motifs and a reduced content of glycine residues as compared with naturally occurring fibroin. Have. The domain sequence of the fourth modified fibroin lacked at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with another amino acid residue. That is, it is a modified fibroin having the characteristics of the above-mentioned second modified fibroin and the third modified fibroin. Specific aspects and the like are as described in the second modified fibroin and the third modified fibroin.

As more specific examples of the fourth modified fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410) ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by. Specific embodiments of the modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

The fifth modified fibroin had its domain sequence replaced with one or more amino acid residues in the REP compared to naturally occurring fibroin, and / or REP. It may have an amino acid sequence containing a region having a large hydrophobic index locally, which corresponds to the insertion of one or a plurality of amino acid residues having a large hydrophobic index.

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

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

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

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

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

For the hydrophobicity index of amino acid residues, a known index (Hydropathic index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic protein, B. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m. (Hereinafter referred to as "sequence A") is used. First, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated for all REPs contained in the sequence A. 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 consecutive 4 amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index", it should be included as one 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 the sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of .6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are duplicated by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 4, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) _n motif existing at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

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

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

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) Is 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 set forth in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666). Alternatively, a modified fibroin containing (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 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 shown in SEQ ID NO: 19 is an amino acid sequence consisting of 3 amino acid residues every other REP, except for the terminal on the C-terminal side, with respect to the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410). VLI) was inserted at two locations, and some glutamine (Q) residues were replaced with serine (S) residues, and some amino acids on the C-terminal side were deleted. The amino acid sequence shown by SEQ ID NO: 20 is the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. is there. The amino acid sequence shown in SEQ ID NO: 21 is the amino acid sequence shown in SEQ ID NO: 8 with two amino acid sequences (VLI) consisting of three amino acid residues inserted every other REP.

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

The modified fibroin of (5-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 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 most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity 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-terminal 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 closest to the C-terminal 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 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, the amino acid sequence set forth in (5-iii) 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 set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 can be mentioned.

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

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

The modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing the 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 most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity 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-terminal 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 closest to the C-terminal 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 according to 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 GPGXX motif in the amino acid sequence of REP.

When the sixth modified fibroin contains the GPGXXX motif in the REP, the content of the GPGXXX motif 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" 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 containing a domain sequence represented by n-} motif (modified fibroin or naturally derived) In (fibroin), the number of GPGXX motifs contained in the region in all REPs included in the sequence 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. Let s be the number obtained by multiplying the total number by 3 (that is, corresponding to 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. The GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluding (A) _{n motifs.}

In the calculation of the GPGXX motif content, "the _{sequence obtained by excluding the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is targeted at "the most C-terminal side". (A) The _{sequence from the n} motif to the C-terminal of the domain sequence (the sequence corresponding to REP) may contain a sequence having a low correlation with the sequence characteristic 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, and this effect is eliminated. When the "GPGXX motif" is located at the C-terminal of the REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

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

The sixth modified fibroin has a glutamine residue content of preferably 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" 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 containing a domain sequence represented by n-} motif (modified fibroin or naturally derived) _{In fibroin), all the sequences from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In the REP of, the total number of glutamine residues contained in the region is u, and the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) _n. The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif. In the calculation of the glutamine residue content, the _{reason why "the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

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

The "other amino acid residue" may be an 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 as shown in Table 1.

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

The sixth modified fibroin has a REP hydrophobicity of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even 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, the "hydrophobicity of REP" is a value calculated by the following method. Formula 1: [(A) _n- motif-REP] _m , or Formula 2: [(A) _n- motif-REP] _m- (A) _{Fibroin containing a domain sequence represented by n-} motif (modified fibroin or naturally derived) _{In fibroin), all the sequences from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In the REP of, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the _{sequence from the (A) n} motif located most on the C-terminal side 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 the _{n motif.} In the calculation of the hydrophobicity of REP, the _{reason for targeting "the sequence 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" is the above-mentioned reason. The same is true.

The sixth modified fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in REP. In addition to the modification corresponding to the substitution of one or more amino acid residues, there may be further modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. ..

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

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

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

The amino acid sequence shown in SEQ ID NO: 30 is obtained by substituting VL for all QQs in the amino acid sequence (Met-PRT525) shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 31 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.

The amino acid sequence shown by SEQ ID NO: 32 is a region of 20 domain sequences existing in the amino acid sequence (Met-PRT410) shown by SEQ ID NO: 7 (however, a few amino acid residues on the C-terminal side of the region are substituted. ) Is repeated twice, all QQs in the sequence are replaced with VF, and the remaining Qs are 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) has SEQ ID NO: 25 and SEQ ID NO: 26.
, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32.

The modified fibroin of (6-ii) is 90% or more of the 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 contains an amino acid sequence having the sequence identity of. The modified fibroin of (6-ii) is also a domain 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 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, visualization and the like of modified fibroin.

As more specific examples of the modified fibroin containing the tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), Modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698) or SEQ ID NO: 40 (PRT966), or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 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 can be mentioned.

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

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

The modified fibroin of (6-iv) is 90% or more of the amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. It contains an amino acid sequence having the sequence identity of. The modified fibroin of (6-iv) is also a domain 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 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. Further, the modified fibroin of (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 according to 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 above-mentioned characteristics.

Since the obtained fibroin molded product has excellent breaking strength, further excellent solvent resistance, and higher fineness when it is fibrous, the modified fibroin has histidine residue, arginine residue, and cysteine. It preferably has at least one amino acid residue selected from the group consisting of residues and serine residues, and the transition metal ion contained in the dope solution is bound to at least a part of the amino acid residues. It is more preferable that the modified fibroin has a histidine residue and the transition metal ion is bound to at least a part of the histidine residue.

The reason why such an effect can be obtained is considered as follows. That is, it is known that metal ions such as copper ion, nickel ion, zinc ion, cobalt ion and iron ion and amino acids such as histidine and cysteine have high affinity and form a coordination bond. For example, it is known that a target protein can be separated by chromatography utilizing the fact that the affinity differs depending on the combination of a metal ion and an amino acid. Specifically, in nickel column affinity chromatography, a strong interaction occurs due to the formation of a coordination bond between nickel ions and cobalt ions and histidine (imidazole ring), which is used as a target. Recover protein. Such separation methods are also used commercially.

Further, "Mozhdehi et.al., Macromolecules, 2016,49,6310-6321, Tuning Dynamic Mechanical Response in Metallopolymer Networks Through Simulultaneous Control of Structural and Temporal Properties of the Networks" in a copolymer having an imidazole group, zinc Ions, copper ions and cobalt ions are allowed to interact with each other to form a metal complex in which these metal ions are coordinated to an imidazole group, and the correlation between the chemical ratio of the ligand and the metal ion and the mechanical properties. The relationship has been reported.

Also in the dope solution according to the present embodiment, the modified fibroin has at least one amino acid residue selected from the group consisting of histidine residue, arginine residue, cysteine residue and serine residue, and is contained in the dope solution. When the contained transition metal ion is bonded to at least a part of the above-mentioned amino acid residue, the obtained fibroin molded product has excellent breaking strength, further excellent solvent resistance, and is fibrous. It is considered that the fineness will be higher.

(Method for Producing Modified Fibroin) The modified fibroin according to the present embodiment is, for example, 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. It can be produced by expressing the nucleic acid in a transformed host.

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding natural fibroin and modifying it by a genetic engineering method, or a method of chemically synthesizing it. be able to. The chemical synthesis method of nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of fibroin obtained from NCBI's web database, etc., AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. Genes can be chemically synthesized by 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 the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added 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 the modified fibroin in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (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 depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector containing a promoter at a position capable of autonomous replication in a host cell, integration into a host chromosome, and transcription of a nucleic acid encoding modified fibroin is preferably used.

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

Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satirus and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Philum and the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, as a vector into which a nucleic acid encoding modified fibroin is introduced, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, Examples thereof include pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569).

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, 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 an expression vector into the host cell, any method for introducing DNA into the host cell can be used. 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 can be mentioned.

As a method for expressing 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 an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

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

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

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture 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 an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

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

The modified fibroin expressed can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer solution, and then an ultrasonic crusher, a French press, or a manton. Crush the host cells with a gaulin homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, methods usually used for isolation and purification of modified fibroin, that is, solvent extraction method, salting-out method by sulfurization, desalting method, organic solvent Anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), and a resin such as S-Sepharose FF (manufactured by Pharmacia). Cationic exchange chromatography method, hydrophobic chromatography method using resins such as butyl Sepharose and phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis such as isoelectric point electrophoresis, etc. Purified preparations can be obtained by using methods such as the law alone or in combination.

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble matter of the modified fibroin is recovered as a precipitate fraction by similarly collecting the host cell, crushing it, and centrifuging it. The insoluble form of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

(Transition metal ion)

The transition metal ion according to the present embodiment is at least one selected from the group consisting of iron ion, ruthenium ion, osmium ion, cobalt ion, nickel ion, copper ion and zinc ion, and is excellent in cost effectiveness. , Iron ion, cobalt ion, nickel ion, copper ion and zinc ion are preferable, cobalt ion, nickel ion and zinc ion are more preferable, and zinc ion is further preferable.

The transition metal ion contained in the dope solution according to the present embodiment is usually blended in the dope solution by dissolving the transition metal salt in formic acid. Transition metal salts include zinc chloride, zinc bromide, zinc acetate, zinc sulfate heptahydrate and zinc acetylacetonate, iron chloride, iron bromide, iron acetate and iron acetylacetonate, nickel acetate, nickel chloride, nickel. Acetylacetonate hydrate and nickel sulfate hexahydrate, cobalt bromide, cobalt acetate and cobalt acetylacetonate dihydrate, and copper chloride, copper bromide, copper acetate and copper acetylacetonate, etc. may be mentioned. it can.

The transition metal salt is preferably zinc chloride, iron bromide, nickel sulfate hexahydrate, nickel acetylacetonate hydrate and cobalt acetate, and zinc chloride and nickel sulfate hexahydrate, because they are more soluble. More preferably, it is a product and cobalt chloride hexahydrate. The transition metal salt may be used alone or in combination of two or more.

The method for preparing the doping solution is not particularly limited, but the doping solution is usually prepared by a method including dissolving modified fibroin, a transition metal salt, and if necessary, other components in formic acid. The dope solution may be agitated or shaken for some time. At that time, the doping liquid may be heated if necessary. For example, the doping solution may be heated to 50 ° C. or higher, 60 ° C. or higher, 70 ° C. or higher, 80 ° C. or higher, 90 ° C. or higher, or 120 ° C. or higher. The upper limit of the heating temperature is not particularly limited, but usually 130 ° C. or lower or about 85 ° C. is sufficient.

The viscosity of the doping liquid is not particularly limited as long as it can be spun, but in consideration of industrial productivity, it is preferably 5,000 to 200,000 mPa · sec, and 10,000 to 180,000 mPa · sec. Is more preferable, 15,000 to 175,000 mPa · sec is even more preferable, and 13,000 to 120,000 mPa · sec may be used.

The content of transition metal ions in the doping liquid according to the present embodiment is 5 mmol / l or more and 100 mmol / l or less, 8 mmol / l or more and 100 mmol / l or less, 10 mmol / l or more and 100 mmol / l or less, based on the total amount of the doped liquid. 10 mmol / l or more and 90 mmol / l or less, 10 mmol / l or more and 80 mmol / l or less, 10 mmol / 70 mmol / l or less, 10 mmol / l or more and 60 mmol / l or less, 12 mmol / l or more and 60 mmol / l or less, 14 mmol / l or more and 60 mmol / l or less Hereinafter, it is preferably 10 mmol / 50 mmol / l or less, 12 mmol / l or more and 50 mmol / l or less, or 14 mmol / 50 mmol / l or less. When the content of the transition metal ion is 5 mmol / l or more, the obtained fibroin molded product has excellent breaking strength, more excellent solvent resistance, and more fineness when it is fibrous. When the content of the transition metal ion is 100 mmol / l or less, it is possible to avoid a decrease in productivity due to a significant increase in viscosity.

The content of the modified fibroin in the doping solution according to the present embodiment is preferably 5 to 40% by weight, more preferably 7 to 40% by weight, when the total amount of the doping solution is 100% by weight. It is more preferably about 40% by weight, more preferably 7 to 35% by weight, more preferably 10 to 35% by weight, and even more preferably 12 to 35% by weight. When the doping solution is used for spinning, the content of the modified fibroin in the doping solution is preferably 15 to 35% by weight, more preferably 15 to 30% by weight, when the total amount of the doping solution is 100% by weight. It is preferably 20 to 35% by weight, more preferably 20 to 30% by weight.

Examples of other components include organic solvents other than formic acid, water and the like.

(Modified fibroin molded product)

The fibroin molded product according to the embodiment of the present invention is a modified fibroin and at least one transition metal selected from the group consisting of iron ion, ruthenium ion, osmium ion, cobalt ion, nickel ion, copper ion and zinc ion. Including with ions. Since the fibroin molded product according to the present embodiment contains the modified gibroin and the transition metal ion, the fibroin molded product according to the present embodiment has excellent solvent resistance and, when it is a fiber, has a high fineness. Further, the fibroin molded product according to the present embodiment has high breaking point strength when it is a fiber. The fibroin molded product may be, for example, a fiber, a film, a gel or a porous body.

The modified fibroin and transition metal ions are as described above. The modified fibroin molded product can be produced by the method for producing a modified fibroin molded product described later.

In the modified fibroin molded product, the total content of transition metal ions may be 5 to 35% by mass, preferably 10 to 35% by mass, preferably 10 to 30% by mass, assuming that the modified fibroin is 100% by mass. 10 to 28% by mass is more preferable, 12 to 28% by mass is further preferable, and 15 to 28% by mass is particularly preferable.

<Manufacturing method of modified fibroin molded product>

An example of a method for producing a modified fibroin molded product will be described below.

The method for producing a modified fibroin molded product according to the present embodiment includes a coagulation step. The coagulation step is a step of removing formic acid from the dope solution according to the above embodiment to obtain a coagulated product of the dope solution. Specifically, the coagulation step may be a step of removing formic acid and coagulating by extruding the dope solution according to the above embodiment into the coagulation liquid, and extruding the dope solution according to the above embodiment into the air. It may be a step of heating formic acid to vaporize and coagulate it. The modified fibroin molded article may be molded into a fiber, film, gel or porous body.

When the modified fibroin molded product is a fiber, it can be produced by a known wet spinning method, dry spinning method, dry wet spinning method, melt spinning method or the like. In the spinning method, for example, it is obtained by a method of removing formic acid from a spun dispersion liquid (thread) by using the doping liquid according to the above embodiment for spinning.

When the modified fibroin molded product is a film, the film is obtained by a method of forming a film of the doping solution according to the above embodiment and removing formic acid from the formed film. A method for producing a film from a fibroin-derived protein is described in International Publication No. 2014/1037799, which is basically obtained according to this method.

When the modified fibroin molded product is a gel, it is obtained by forming a gel of a dope solution containing the modified fibroin, a transition metal ion, and formic acid, and removing formic acid from the gel. A method for producing a gel from a fibroin-derived protein is described in International Publication No. 2014/175177, which is basically obtained according to this method.

When the modified fibroin molded product is a porous body, a method for producing the porous body from a fibroin-derived protein is described in International Publication No. 2014/175178, which is basically obtained by this method.

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

<Example 1 and Comparative Example 1>

(1) Manufacture of modified fibroin

(1-1) Preparation of expression vector

A modified fibroin having SEQ ID NO: 40 (hereinafter, also referred to as "PRT966") based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes. , A modified fibroin having SEQ ID NO: 15 (hereinafter, also referred to as “PRT799”) and a modified fibroin having SEQ ID NO: 37 (hereinafter, also referred to as “PRT918”) were designed. The amino acid sequence shown by SEQ ID NO: 40 includes all QQs in the sequence in which the regions of the 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 are repeated twice for the purpose of improving the hydrophobicity. It has an amino acid sequence in which VF is substituted and the remaining Q is substituted with I, and the amino acid sequence shown by SEQ ID NO: 11 is further added to the N-terminal. Further, the amino acid sequence shown by SEQ ID NO: 15 has an amino acid sequence in which amino acid residues are substituted, inserted or deleted for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes. Further, the amino acid sequence (tag sequence and hinge sequence) shown by SEQ ID NO: 12 is added to the N-terminal.

Next, nucleic acids encoding artificial structural proteins (modified fibroin) PRT966, PRT799 and PRT918 having the designed amino acid sequence were synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.

(1-2) Expression of modified fibroin

Escherichia coli BLR (DE3) was transformed with the expression vector obtained in (1-1) above. 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 OD _{600 was 0.005.} The culture solution temperature was maintained at 30 ° C., _{and flask culture was carried out 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 cells were cultured under constant pH 6.9. Further, 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 cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of modified fibroin. Twenty hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture medium before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of the desired modified fibroin-sized band depending on the addition of IPTG. did.

(1-3) Purification of modified fibroin

The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The 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) to a concentration of 100 mg / mL at 60 ° C. Was stirred with a stirrer for 30 minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white agglutinating protein obtained after dialysis was recovered by centrifugation, water was removed by a freeze-dryer, and the freeze-dried powder was recovered to obtain modified fibroin (modified spider fibroin of PRT966, PRT799 and PRT918). ..

(2) Preparation of doping solution

Formic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and zinc chloride (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) were mixed as a solvent and stirred at room temperature for 1 hour. Next, the modified fibroin (PRT966) obtained in the above production step was added, and the mixture was stirred and dissolved at room temperature for 2 hours to prepare a doping solution. The solvent, the transition metal salt and the modified fibroin were blended so that the contents of the transition metal salt and the modified fibroin with respect to the total amount of the dope solution had the values shown in Table 6. Table 6 shows the transition metal ion content [mmol / l] and the viscosity of the doping solution with respect to the total amount of the prepared doping solution.

(3) Evaluation of stability of doping solution

The obtained dope solution was evaluated according to the following evaluation criteria. The results are shown in Table 6.

A: Does not show gel after being left at room temperature for 24 hours.

C: Gels appear after being left at room temperature for 24 hours

(4) Manufacture and evaluation of modified fibroin molded product

(4-1) Production of modified fibroin molded product (spider silk fibroin fiber)

The dope liquid prepared above was filled in a reserve tank, and the dope liquid was discharged from a spinning nozzle (spinning cap) in a coagulation bath using a gear pump to form a raw yarn (thread). Then, the coagulated raw yarn was stretched in a water washing bath. After washing and stretching in a water washing bath, the mixture was dried using a drying plate, and the obtained spider silk fibroin molded product (fiber) was wound up with a winder. The conditions for wet spinning were as follows. The total draw ratio was 5 times.

Hole diameter of spinneret: 0.2 mm

Coagulant: 100% methanol

Coagulant temperature: room temperature

Water wash bath temperature: 60 ° C

Stretching bath temperature: 60 ° C

Total draw ratio: 3.5 times or 5 times

Drying temperature: 60 ° C

(4-2) Evaluation of mechanical properties of modified fibroin molded product

The mechanical properties of the molded product (fiber) obtained in (4-1) above were measured by the following method. The fineness and breaking strength of 15 randomly sampled fibers were measured using "FAVIMAT", a single yarn strength and elongation measuring device manufactured by Textechno, in an environment of a temperature of 20 ° C. and a relative humidity of 65% (sample). Number = 15), the average value was calculated. The measurement conditions for the breaking point strength were a load cell capacity of 210 cN, a gauge length of 20 mm, and a tensile speed of 10 mm / min. The results are shown in Table 6. The relative values of breaking strength and fineness are values when the values of breaking strength and fineness measured in Comparative Example 1 described later are set to 100.

(4-3) Solvent resistance test

The solvent resistance test of the modified fibroin molded product (fiber) was carried out as follows. That is, the modified fibroin molded product obtained in (4-1) above was added to HFIP (dissolving solvent) contained in a container so as to have a concentration of 1 mg / ml, and the time required for dissolution was observed. The observation was performed visually. The results are shown in Table 6.

(5) Measurement of molecular weight

The solution obtained by dissolving the modified fibroin molded product obtained in (4-1) above in HFIP is filtered through a filter having a pore size of 0.45 μm, and then subjected to HPLC gel permeation chromatography (GPC) analysis to obtain a number average molecular weight (Mn). , Weight average molecular weight (Mw), peak top molecular weight (Mp) and dispersity (Mw / Mn) were determined. The number average molecular weight, weight average molecular weight and peak top molecular weight were measured using a standard polystyrene calibration curve. The measurement conditions using polystyrene were as follows. The results are shown in Table 6.

Guard column: Shodex GPC HFIP-606M (2)

Analytical column: Shodex GPC HFIP-G-4A

Flow velocity: 0.4 ml / min

Carrier: HFIP containing 1-2 mM sodium trifluoroacetate

<Comparative example 1>

After producing the modified fibroin in the same manner as in Example 1, the dope solution was adjusted in the same manner as in Example 1 except that the transition metal salt was not added. The stability of the obtained doped liquid was evaluated in the same manner as in Example 1. Further, the obtained dope solution was produced and evaluated as a modified fibroin molded product in the same manner as in Example 1, and the molecular weight was measured. The results are shown in Table 6.

As shown in Table 6, the dope solution containing the transition metal ion (zinc ion) (Example 1) has an increased viscosity of the dope solution as compared with the dope solution not containing the transition metal ion (Comparative Example 1). Was there. The increase in the viscosity of the dope solution suggested that the transition metal ion and the amino acid residue in the modified fibroin interacted in the dope solution to form a coordination bond.

Further, as shown in Table 6, in the modified fibroin molded article (Example 1) produced using a dope solution containing a transition metal ion, a molded article produced using a dope solution containing no transition metal ion (Comparative Example). Compared with 1), the fineness and breaking strength were improved.

Further, in the molded body containing no transition metal ion (Comparative Example 1), when the molded body was added to HFIP, it was immediately dissolved, whereas in the modified fibroin molded body of Example 1 containing transition metal ion, the molded body was added to HFIP. After the addition, it took 5 minutes to dissolve, and the solvent resistance was improved.

The reason why the solvent resistance is improved when the dope solution contains transition metal ions is that in the modified fibroin molded product produced by using the modified fibroin and the dope solution containing the transition metal ion, the amino acid residue of the modified fibroin is histidine. It is considered that this is because the transition metal ion and the transition metal ion form a coordination bond.

Further, as shown in Table 6, the molecular weight of the modified fibroin molded product containing the transition metal ion (Example 1) is the same as the molecular weight of the molded product not containing the transition metal ion (Comparative Example 1), and a difference is observed. I couldn't. This is because the zinc ion is insoluble in HFIP used as a solvent, or the coordination bond between the zinc ion and the amino acid residue in the modified fibroin is not maintained in the HFIP solution. It is presumed that it could not be seen.

<Examples 2 and 3>

After producing the modified fibroin in the same manner as in Example 1, the solvent, zinc chloride and the modified fibroin were blended so that the contents of the zinc ion and the modified fibroin with respect to the total amount of the dope solution were the values shown in Table 7. Prepared the dope solution in the same manner as in Example 1 and evaluated the stability. Further, a modified fibroin molded product was produced and evaluated in the same manner as in Example 1 except that the total draw ratio was 3.5 times. The results are shown in Table 7. The relative values of breaking strength and fineness are values when the values of breaking strength and fineness of the modified fibroin molded product measured in Comparative Example 2 described later are set to 100. Table 7 also shows the transition metal ion content [mmol / l] and the viscosity of the doping solution with respect to the total amount of the prepared doping solution.

<Comparative example 2>

After producing the modified fibroin in the same manner as in Example 2, the dope solution was prepared in the same manner as in Example 2 except that the transition metal salt was not added, and the stability was evaluated. Moreover, the modified fibroin molded article was produced and evaluated in the same manner as in Example 2. The results are shown in Table 7.

<Comparative example 3>

After producing the modified fibroin in the same manner as in Example 3, the dope solution was prepared in the same manner as in Example 3 except that dimethyl sulfoxide (DMSO) was used as a solvent, and the stability was evaluated. The results are shown in Table 7.

As shown in Table 7, even when the content of the modified fibroin is 28% by mass, the dope solution containing the transition metal ion (zinc ion) (Examples 2 and 3) is the dope solution containing no transition metal ion (Examples 2 and 3). Compared with Comparative Example 2), the viscosity of the dope solution was increased. The increase in the viscosity of the dope solution suggested that the transition metal ion and the amino acid residue formed a coordination bond. In addition, when DMSO is used as the solvent (Comparative Example 3), it immediately becomes gel-like when metal ions are added, and the gel state is maintained even when heated to a high temperature, and the viscosity of the dope solution can be measured. There wasn't. As described above, the doping solution using DMSO as a solvent lacked stability and was unsuitable as a doping solution.

Further, as shown in Table 7, even when the total draw ratio is 3.5 times, the transition metal ions are produced in the modified fibroin molded product (Examples 2 and 3) produced by using the dope solution containing the transition metal ions. The fineness and breaking strength were improved as compared with the molded product (Comparative Example 2) produced by using the dope solution containing no.

<Examples 4 and 5>

After producing the modified fibroin in the same manner as in Example 1, nickel sulfate hexahydrate (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was used as the transition metal salt in place of zinc chloride in Example 4, and in Example 5. Zinc chloride hexahydrate (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added, and the contents of the transition metal salt and the modified fibroin relative to the total amount of the dope solution were the values shown in Table 8, respectively. The dope solution was prepared in the same manner as in Example 1 except that the salt and the modified fibroin were blended, and the stability was evaluated. The results are shown in Table 8. Table 8 also shows the transition metal ion content [mmol / l] and the viscosity of the doping solution with respect to the total amount of the prepared doping solution.

<Comparative example 4>

After producing the modified fibroin in the same manner as in Example 4, the dope solution was prepared in the same manner as in Example 4 except that the transition metal salt was not added, and the stability was evaluated. The results are shown in Table 8.

As shown in Table 8, even when the transition metal ion contained in the dope solution is nickel ion (Example 4) or cobalt ion (Example 5), the dope solution does not contain the transition metal ion (Comparative Example). Compared with 4), the viscosity of the dope solution was increased. The increase in the viscosity of the dope solution suggested that the transition metal ion and the amino acid residue formed a coordination bond.

Reference Example 1: Combustibility test of modified fibroin

A lyophilized powder of modified fibroin (PRT799) was added to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) to a concentration of 24% by mass, and the mixture was mixed for 3 hours using a shaker. It was dissolved. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The obtained spinning stock solution is heated to 90 ° C., filtered through a metal filter having a mesh size of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100 mass from a solid nozzle having a needle diameter of 0.2 mm. % Methanol was discharged into a coagulation bath. The discharge temperature was 90 ° C. After solidification, the obtained raw yarn was wound up and air-dried to obtain modified fibroin fiber (raw material fiber).

A knitted fabric (thickness: 180 denier, gauge number: 18) was produced by circular knitting using a circular knitting machine using twisted yarn obtained by twisting raw material fibers. 20 g of the obtained knitted fabric was cut out and used as a test piece.

The flammability test was based on the "test method for synthetic resin having a fine powder or a low melting point" described in "Fire Danger No. 50 (dated May 31, 1995)". The test was carried out under the conditions of a temperature of 22 ° C., a relative humidity of 45% and an atmospheric pressure of 1021 hPa. Table 9 shows the measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)).

As a result of the flammability test, the critical oxygen index (LOI) value of the knitted fabric knitted with the modified fibroin (PRT799) fiber was 27.2. Generally, when the LOI value is 26 or more, it is known to be flame-retardant. It can be seen that the modified fibroin is excellent in flame retardancy. As is clear from this, the critical oxygen index (LOI) value of the molded product such as fiber or resin produced by using the modified fibroin used in the present invention is preferably 26 or more.

Reference example 2: Evaluation of heat absorption and heat generation of modified fibroin

A lyophilized powder of modified fibroin was added to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) to a concentration of 24% by mass, and the mixture was dissolved by mixing for 3 hours using a shaker. .. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The obtained spinning stock solution is heated to 60 ° C., filtered through a metal filter having a mesh size of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100 mass from a solid nozzle having a needle diameter of 0.2 mm. % Methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C. After solidification, the obtained raw yarn was wound up and air-dried to obtain modified fibroin fiber (raw material fiber).

For comparison, commercially available wool fibers, cotton fibers, tencel fibers, rayon fibers and polyester fibers were prepared as raw material fibers.

Each knitted fabric was produced by weft knitting using a weft knitting machine using each raw material fiber. Table 10 shows the thickness and the number of gauges of the knitted fabric using PRT918 fiber or PRT799 fiber. The thickness and the number of gauges of the knitted fabric using the other raw material fibers were adjusted so as to have almost the same coverage factor as the knitted fabric of the modified fibroin fiber. Specifically, it is as follows.

Two knitted fabrics cut into 10 cm × 10 cm were put together, and the four sides were sewn together to form a test piece (sample). The test piece is left in a low humidity environment (temperature 20 ± 2 ° C., relative humidity 40 ± 5%) for 4 hours or more, and then transferred to a high humidity environment (temperature 20 ± 2 ° C., relative humidity 90 ± 5%). The temperature was measured at 1-minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.

From the measurement results, the maximum degree of heat absorption and heat generation was determined according to the following formula A.

Formula A: Maximum heat absorption and heat generation = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then moved to a high humidity environment)-(Sample, sample Sample temperature when moving to a high humidity environment after being placed in a low humidity environment until the temperature reaches equilibrium)} (° C) / sample weight (g)

FIG. 6 is a graph showing an example of the results of the heat absorption and heat generation test. The horizontal axis of the graph shows the time (minutes) left in the high humidity environment, where 0 is the time when the sample is moved from the low humidity environment to the high humidity environment. The vertical axis of the graph shows the temperature (sample temperature) measured by the temperature sensor. In the graph shown in FIG. 6, the point indicated by M corresponds to the maximum value of the sample temperature.

Table 11 shows the calculation results of the maximum heat absorption and heat generation of each knitted fabric.

As shown in Table 11, it can be seen that the modified fibroin (PRT918 and PRT799) has a higher maximum degree of heat absorption and heat generation and is excellent in heat absorption and heat generation as compared with the existing materials. As is clear from such a fact, a molded product such as a fiber produced by using the modified fibroin used in the present invention generally has a maximum heat absorption and heat generation degree of more than 0.025 ° C./g obtained according to the above formula A. is there.

Reference example 3: Evaluation of heat retention of modified fibroin

A lyophilized powder of modified fibroin was added to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) to a concentration of 24% by mass, and the mixture was dissolved by mixing for 3 hours using a shaker. .. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

For comparison, commercially available wool fibers, silk fibers, cotton fibers, rayon fibers and polyester fibers were prepared as raw material fibers.

Each knitted fabric was produced by weft knitting using a weft knitting machine using each raw material fiber. The count, number of twists, number of gauges, and basis weight of the knitted fabric using PRT966 fiber or PRT799 fiber are as shown in Table 12. The knitted fabric using other raw material fibers was adjusted so as to have almost the same coverage factor as the knitted fabric of the modified fibroin fiber. Specifically, it is as follows.

The heat retention was evaluated by using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd. and using a dry contact method (a method assuming direct contact between the skin and clothes in a dry state). One knitted fabric cut into a rectangle of 20 cm × 20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) dissipated through the test piece was determined under the condition of a wind speed of 30 cm / sec in the wind tunnel. The amount of heat (b) dissipated under the same conditions as above was determined without setting the test piece, and the heat retention rate (%) was calculated according to the following formula B.

Formula B: Heat retention rate (%) = (1-a / b) x 100

From the measurement results, the heat retention index was calculated according to the following formula C.

Formula C: Heat retention index = heat retention rate (%) / sample basis weight (g / m ² )

The calculation results of the heat retention index are shown in Table 13. The higher the heat retention index, the more excellent the heat retention material can be evaluated.

As shown in Table 13, it can be seen that the modified fibroin (PRT966 and PRT799) has a higher heat retention index and is excellent in heat retention as compared with the existing materials.

As shown in Reference Examples 1 to 3, when the modified fibroin is the modified spider silk fibroin, it can be made more excellent in heat retention, heat absorption and heat generation and / or flame retardancy, and is excellent in solvent resistance and fineness. Fiber can be obtained.

Claims

A dope solution containing modified fibroin, at least one transition metal ion selected from the group consisting of iron ion, ruthenium ion, osmium ion, cobalt ion, nickel ion, copper ion and zinc ion, and formic acid.
The doping solution according to claim 1, wherein the modified fibroin is selected from the group consisting of modified silk fibroin and modified spider silk fibroin.
The dope solution according to claim 1 or 2, wherein the transition metal ion is selected from the group consisting of cobalt ion, nickel ion and zinc ion.
The doping solution according to any one of claims 1 to 3, wherein the transition metal ion is a zinc ion.
The doping solution according to any one of claims 1 to 4, wherein the modified fibroin is modified spider silk fibroin.
The modified fibroin has at least one amino acid residue selected from the group consisting of histidine residue, arginine residue, cysteine residue and serine residue, and the transition metal ion is at least the amino acid residue. The dope solution according to any one of claims 1 to 5, which is partially bound.
The dope solution according to claim 6, wherein the modified fibroin has a histidine residue, and the transition metal ion is bound to at least a part of the histidine residue.
A method for producing a modified fibroin molded product, which comprises a coagulation step of removing the formic acid from the dope solution according to any one of claims 1 to 7 to obtain a coagulated product of the dope solution.
The production method according to claim 8, wherein the coagulation step is a step of discharging the doping liquid into the coagulation liquid to coagulate it.
The production method according to claim 8 or 9, wherein the modified fibroin molded product is a fiber or film containing the modified fibroin.