WO2019151430A1

WO2019151430A1 - Protein fiber yarn, woven body, method for manufacturing protein fiber yarn, and method for manufacturing woven body

Info

Publication number: WO2019151430A1
Application number: PCT/JP2019/003465
Authority: WO
Inventors: 昌三鳥越; 下田　誠治; 明彦尾関
Original assignee: 株式会社島精機製作所; Ｓｐｉｂｅｒ株式会社
Priority date: 2018-01-31
Filing date: 2019-01-31
Publication date: 2019-08-08
Also published as: JP2021055191A

Abstract

A protein fiber yarn, which is a single yarn that is one of a twist yarn containing modified fibroin fibers, a false twist yarn containing modified fibroin fibers, and a spun yarn containing modified fibroin fibers, wherein the moisture content thereof is reduced after being twisted.

Description

Protein fiber yarn and woven fabric, protein fiber yarn manufacturing method and woven fabric manufacturing method

The present invention relates to a protein fiber yarn and a knitted fabric having a twisting history, and a protein fiber yarn manufacturing method and a knitted fabric manufacturing method.

As is well known, when yarns having a history of twisting, such as spun yarns, twisted yarns, and false twisted yarns, are knitted using a single yarn, skewing often occurs. This is considered because residual torque resulting from twisting is generated in the yarn. Therefore, for example, when knitting a spun yarn, generally a single twisted yarn is twisted in the opposite direction to a twin yarn to reduce the residual torque caused by the lower twist. Yes. By using this double yarn, the skew is eliminated. However, in order to obtain a twin yarn, it is necessary to prepare a yarn with a fine count which is half of the target yarn count, and it takes time to produce the fine count yarn. In addition, the low-definition fiber necessary for obtaining a fine count yarn requires a high level of technology for its production and increases the cost.

On the other hand, when producing a woven fabric, a single yarn is generally used for both warp and weft. For this reason, even in the woven fabric, distortion due to the residual torque of the single yarn may occur. Further, one of the troubles of textile products is the occurrence of pilling. Pilling, that is, fluff, can be produced by fuzz generated on yarns constituting woven fabrics or knitted fabrics moving by friction and entangled with each other. When the residual torque of the yarn increases, the length, amount, etc. of the yarn causing the pilling increases by an amount corresponding thereto. The length and amount of such fluff greatly affects the amount of pilling. Although drugs capable of suppressing pilling are known, it cannot be said that it is sufficient in terms of environmental burden.

JP 2006-52493 A

The present invention provides a protein fiber yarn and a method for producing the same, in which skewing in the knitted fabric, distortion in the woven fabric, and pilling in the knitted fabric and the woven fabric are less likely to occur, and a knitted fabric using the protein fiber yarn and A manufacturing method thereof is provided.

The present invention relates to the following [1] to [9].
[1] A single yarn that is one of a twisted yarn containing a modified fibroin fiber, a false twisted yarn containing a modified fibroin fiber, and a spun yarn containing a modified fibroin fiber. Reduced protein fiber yarn.
[2] A single yarn of the spun yarn containing the modified fibroin fiber, which is exposed to a first humidity environment in at least a twisting step for twisting the modified fibroin fiber, and the first yarn after the twisting step. The protein fiber yarn according to [1], wherein the moisture content is reduced by being exposed to a second humidity environment having a humidity lower than that of the first humidity environment.
[3] The protein fiber yarn according to [1] or [2], wherein the modified fibroin is a modified spider silk fibroin.
[4] A knitted fabric obtained by knitting or weaving the protein fiber yarn according to any one of [1] to [3].
[5] A method for producing a protein fiber yarn, which produces a single yarn that is one of a twisted yarn containing a modified fibroin fiber, a false twisted yarn containing a modified fibroin fiber, and a spun yarn containing the modified fibroin fiber. A protein fiber yarn comprising: a twisting step of obtaining the single yarn by twisting a fiber containing fibroin fiber; and a moisture content lowering step that is performed after the twisting step and reduces the moisture content of the single yarn. Manufacturing method.
[6] A spinning step of spinning a single yarn of a spun yarn containing the modified fibroin fiber, the spinning step including the twisting step of twisting the modified fibroin fiber in a first humidity environment, and the spinning The moisture content lowering step, which is performed after the step and reduces the moisture content of the single yarn under a second humidity environment having a lower humidity than the first humidity environment, according to [5]. A method for producing a protein fiber yarn.
[7] The humidity under the first humidity environment is 60% or more, the humidity under the second humidity environment is 50% or less, and the moisture content of the single yarn is 15% in the moisture content lowering step. The method for producing a protein fiber yarn according to [6], which is reduced to the following (in terms of official moisture content).
[8] The method for producing a protein fiber yarn according to any one of [5] to [7], wherein the modified fibroin is a modified spider silk fibroin.
[9] A method for producing a knitted fabric, wherein the single yarn produced by the method for producing a protein fiber yarn according to any one of [5] to [8] is knitted or woven.

According to the present invention, for example, the moisture content after the cone-up of the protein fiber yarn is lowered than the moisture content in the process before the cone-up including at least the twisting process, thereby causing skewing and distortion. The obtained residual torque can be eliminated. In addition, we do not use harmful chemicals because we find the final effect by controlling moisture in the fiber. Therefore, the present invention is excellent in terms of environmental load and productivity. Since it is possible to use a single yarn, for example, spinning can be performed with a target count that is twice that of normal. As a result, productivity is improved. Combined yarn processing is unnecessary.

When the knitted fabric is a knitted fabric, there is an advantage that the knitted fabric can be thinned because skewing does not occur when using a single yarn. Therefore, inexpensive products can be provided. When the knitted fabric is a woven fabric, there is no distortion even when using a single yarn, so a beautiful silhouette can be provided. Furthermore, since the protein fiber yarn according to the present invention has a small residual torque, it is difficult for fluff to occur. Therefore, the occurrence of pilling can be suppressed. As a result, soft texture yarns and products can be provided.

FIG. 1 is a schematic diagram showing an example of a modified fibroin domain sequence. FIG. 2 is a schematic diagram showing an example of a modified fibroin domain sequence. FIG. 3 is a schematic diagram showing an example of a modified fibroin domain sequence. FIG. 4 is an explanatory view schematically showing an example of a spinning device for producing a modified spider silk fibroin fiber. FIG. 5 is a photograph of the knitted fabric of Example 1. FIG. 6 is a photograph of the knitted fabric of Comparative Example 1.

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

[Protein fiber yarn and knitted fabric]
The protein fiber yarn according to the present embodiment is a single yarn that is one of a twisted yarn containing a modified fibroin fiber, a false twisted yarn containing a modified fibroin fiber, and a spun yarn containing a modified fibroin fiber, After being applied, the moisture content was lowered. In this specification, the protein fiber yarn means a spun yarn and a twisted yarn made of a twisted yarn, a false twisted yarn that has been twisted in the manufacturing process, and the like. In other words, the protein fiber yarn is a yarn having a history of twisting that causes residual torque. The knitted fabric according to the present embodiment is formed by knitting or weaving the protein fiber yarn according to the present embodiment.

[Producing method of protein fiber yarn and producing method of knitted fabric]
The method for producing a protein fiber yarn according to the present embodiment includes a protein that produces a single yarn that is one of a twisted yarn containing a modified fibroin fiber, a false twisted yarn containing a modified fibroin fiber, and a spun yarn containing the modified fibroin fiber. A method for producing a fiber yarn, a twisting step for obtaining the single yarn by twisting a fiber containing a modified fibroin fiber, and a moisture content which is implemented after the twisting step and reduces the moisture content of the single yarn A lowering step. The method for producing a knitted fabric according to the present embodiment is a method for producing the single yarn by the method for producing a protein fiber yarn according to the present embodiment, and knitting or weaving the produced single yarn.

This embodiment includes a moisture changing step after spinning, crimping, blending, spinning, and cone-up of protein fiber yarn.

<Modified fibroin>
The modified fibroin according to the present embodiment has a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing. In the modified fibroin, an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to these, but are typically regions having no amino acid motif repeat characteristic of fibroin and consisting of about 100 amino acids.

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

As long as the “modified fibroin” has the amino acid sequence specified in the present embodiment, the amino acid sequence of naturally-occurring fibroin may be used as it is, and it depends on the amino acid sequence of naturally-occurring fibroin. The amino acid sequence may be modified (for example, the amino acid sequence may be modified by modifying the gene sequence of a naturally-derived fibroin that has been cloned), or it may be artificially designed without relying on the naturally-occurring fibroin. And those synthesized (for example, those having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

In the present specification, the “domain sequence” refers to a fibroin-specific crystal region (typically corresponding to the (A) _n motif in the amino acid sequence) and an amorphous region (typically in the REP of the amino acid sequence). Amino acid sequence that gives rise to the following formula: Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) amino acid represented by _n motif Means an array. Here, (A) _n motif represents an amino acid sequence mainly composed of alanine residues, and n is 2 to 27. n may be an integer from 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. In addition, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) _n motif may be 40% or more, such as 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 alone. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. Plural REPs may have the same amino acid sequence or different amino acid sequences.

The modified fibroin is, for example, a modification of the amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned natural fibroin gene sequence. Can be obtained at 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-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.

Naturally-derived fibroin is a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. Specific examples include fibroin produced by insects or spiders.

Examples of fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandaraina), Tengea (Antheraea yamanai), 柞蚕 (Antereaperanii), 楓蚕 (Eriothyraminey) ), Silkworms produced by silkworms, such as Samia cythia, chestnut worms (Caligula japonica), Chuser moth (Antherea mylitta), Antheraea assama, and vespax (Vespaxia spp.) Hornet silk protein.

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

Fibroin produced by spiders includes, for example, spiders belonging to the genus spider (Araneus spp.) Such as the spider spider, the spider spider, the red spider spider, and the bean spider, the genus spiders of the genus Araneus, the spider spider spider, the spider spider genus e Spiders, spiders such as spiders, spiders belonging to the genus Spider, spiders belonging to the genus Pronos, spiders belonging to the genus Trinofunda, such as Torinofundamas (genus Cyrtarachne) Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Spider (Ordgarius genus), such as the spiders, the spiders, and the spiders belonging to the genus Ordgarius Spiders belonging to the genus Argiope, such as the genus Argiope, spiders belonging to the genus Arachnura, such as the white-tailed spider, spiders belonging to the genus Acusilas such as the common spider, the spider belonging to the genus Acusilas, and the genus Spider Spiders belonging to (genus Cytophora), spiders belonging to the genus Spider belonging to the genus Spider (genus Poltys) such as spiders, genus Spider, spiders belonging to the genus Spider belonging to the genus Cyclosa (genus Cyclosa), and spiders belonging to the genus Cyclosa (genus Cyclosa) Spider silk proteins produced by spiders belonging to the genus Chorizopes), and Asina, such as Asagaaga spider, Yasagata Asaga spider, Harabiro Ashida spider and Urokoa Asaga spider Spiders belonging to the genus Tetragnata, spiders belonging to the genus Spider genus (Leucage sp.) Such as the white spider spider and the white spider spider, the spider genus belonging to the spider spider genus (Nephila spp. Spiders belonging to the genus Azumigumi (Menosira), spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the common spider spider, the black spider spider, the genus Spider genus belonging to the genus Spider belonging to the genus (L) and the genus Spider belonging to the genus Usd Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops Examples include spider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spiroin 1 [derived from Nephila clavipes] (GenBank accession number 4) ), U37520 (base sequence)), major ampulate spidro n 1 [derived from Latroductus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidolin 2 [derived from Nephila clavata (GenBank accession number AAL32 base sequence 44 AAL32 base sequence amino acid 44, amino acid sequence 44 AAL47) )), Major ampulerate spiroin 1 [from Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major amplospiroid 2 [Euprostenaplas ] (GenBank accession number CAM322249.1 (amino acid sequence), AM490169 (base sequence)), minor sample silk protein 1 [Nephila clubs] (GenBank accession number AAC145891 (amino acid sequence)), minor sample 2 Nephila clavies] (GenBank accession number AAC14591.1 (amino acid sequence)), minor sample spirodin-like protein [Nephilegenes cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), etc.

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

The modified fibroin may be modified silk fibroin (modified silk protein amino acid sequence produced by silkworm), modified spider silk fibroin (modified spider silk protein amino acid sequence produced by spiders) Thing). Among them, modified spider silk fibroin is preferably used.

Specific examples of the modified fibroin include a modified fibroin derived from a large sphincter bookmark silk protein produced in a spider large bottle gland, a modified fibroin with a reduced content of glycine residues, (A) an _n motif Modified fibroin with reduced content, content of glycine residue, and (A) modified fibroin with reduced content of _n motif.

Examples of the modified fibroin derived from the large sphincter bookmark silk protein produced in the spider large bottle gland include a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In the modified fibroin derived from the large sphincter bookmark silk protein produced in the spider large bottle-like gland, in formula 1, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, and more preferably 8 to 20. An integer is further preferred, an integer of 10 to 20 is still more preferred, an integer of 4 to 16 is still more preferred, an integer of 8 to 16 is particularly preferred, and an integer of 10 to 16 is most preferred. In the modified fibroin derived from the large sphincter bookmark silk protein produced in the spider large bottle-like gland, in Formula 1, the number of amino acid residues constituting REP is preferably 10 to 200 residues. More preferably, it is ˜150 residues, more preferably 20-100 residues, and even more preferably 20-75 residues. A modified fibroin derived from the large sphincter bookmark silk protein produced in the spider large bottle gland is a glycine residue contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m , The total number of residues of serine residues and alanine residues is preferably 40% or more, more preferably 60% or more, still more preferably 70% or more based on the total number of amino acid residues. .

The modified fibroin derived from the large sphincter bookmark silk protein produced in the spider large bottle gland comprises a unit of an amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m and has a C-terminal. It may be a polypeptide whose sequence is the amino acid sequence shown in any of SEQ ID NOs: 14 to 16 or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 14 to 16.

The amino acid sequence shown in SEQ ID NO: 14 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 15 is the sequence The amino acid sequence shown in SEQ ID NO: 14 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus, and the amino acid sequence shown in SEQ ID NO: 16 is 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID NO: 14. It is identical to the amino acid sequence.

As a more specific example of a modified fibroin derived from a large sphincter bookmark silk protein produced in the spider large bottle-like gland, (1-i) the amino acid sequence represented by SEQ ID NO: 17, or (1-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence indicated by number 17. The sequence identity is preferably 95% or more.

The amino acid sequence represented by SEQ ID NO: 17 is an amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 18) consisting of a start codon, His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminus. The 13th repeat region was increased to approximately double, and the translation was mutated to terminate at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 17 is identical to the amino acid sequence shown in SEQ ID NO: 16.

The modified fibroin (1-i) may be composed of the amino acid sequence represented by SEQ ID NO: 17.

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

Modified fibroin with a reduced content of glycine residues has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, X Is an amino acid residue other than glycine.) In at least one motif sequence selected from (1), this corresponds to substitution of one glycine residue in at least one or more of the motif sequences with another amino acid residue. It may have an amino acid sequence.

In the modified fibroin with a reduced content of glycine residues, the ratio of the motif sequence in which the above glycine residue is replaced with another amino acid residue may be 10% or more with respect to the total motif sequence. .

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

The modified fibroin in which the content of glycine residues is reduced is that 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. preferable. In the modified fibroin in which the content of glycine residues is reduced, 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, and more preferably 10% or less. More preferably, it is 6% or less, still more preferably 4% or less, still more preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.

The method for calculating z / w will be described in more detail. First, in a fibroin (modified fibroin or naturally-occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , (A) _n located closest to the C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs included 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. Also, for example, when there is an X (center X) included in two XGXs as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, it is 5 amino acid residues). is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the domain sequence to the most C-terminal (A) _n motif to the C-terminus of the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A) _n motif located closest to the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

In the modified fibroin with a reduced content of glycine residues, z / w is preferably 50.9% or more, more preferably 56.1% or more, and 58.7% or more. Is more preferably 70% or more, still more preferably 80% or more. Although there is no restriction | limiting in particular in the upper limit of z / w, For example, 95% or less may be sufficient.

A modified fibroin with a reduced content of glycine residues, for example, encodes another amino acid residue by substituting at least a part of the base sequence encoding the glycine residue from the cloned gene sequence of naturally occurring fibroin. It can obtain by modifying so that. At this time, one glycine residue in GGX motif and GPGXX motif may be selected as a glycine residue to be modified, or substitution may be performed so that z / w is 50.9% or more. In addition, for example, an amino acid sequence satisfying the above-described aspect can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis. In any case, in addition to the modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. The amino acid sequence corresponding to the insertion and / or addition may be modified.

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

As more specific examples of modified fibroin with a reduced content of glycine residues, (2-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2- ii) A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 can be mentioned.

The modified fibroin (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin. The amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence represented by SEQ ID NO: 3, in which every two (A) _n motifs are deleted from the N-terminal side to the C-terminal side, and further before the C-terminal sequence. One [(A) _n motif-REP] is inserted into the. The amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.

The value of z / w in the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 12 are 58.7%, 70.1%, 66.1% and 70.0%. In addition, the x / y values of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 10 and 12 at a jagged ratio (described later) of 1: 1.8 to 11.3 are 15.0% and 15. 0%, 93.4%, 92.7% and 89.3%.

The modified fibroin (2-i) may be composed of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin (2-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.

The above-mentioned modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.

Examples of tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules. Specific examples of the affinity tag include a histidine tag (His tag). The His tag is a short peptide with about 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography) Can be used. Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 5 (amino acid sequence including a His tag sequence and a hinge sequence).

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” using 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. As the epitope tag, HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like can be mentioned. By using the epitope tag, the modified fibroin can be easily purified with high specificity.

Furthermore, a tag sequence that can be separated with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with protease, the modified fibroin from which the tag sequence has been separated can also be recovered.

More specific examples of the modified fibroin containing the tag sequence include (2-iii) the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 8 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The amino acid sequences represented by SEQ ID NOs: 6, 7, 8, 9, 11, and 13 are the amino acids represented by SEQ ID NO: 5 at the N-terminus of the amino acid sequences represented by SEQ ID NOs: 1, 2, 3, 4, 10, and 12, respectively. A sequence (including a His tag sequence and a hinge sequence) is added.

(2-iii) The modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The modified fibroin (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin (2-iv) has an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 with a sequence identity of 90% or more, and is contained in XREP ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.

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

(A) modified fibroin content of _n motifs has been reduced, the domain sequence is compared to the naturally occurring fibroin, having an amino acid sequence reduced the content of (A) _n motif. It can be said that the domain sequence of the modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) _n motifs as compared to naturally occurring fibroin.

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

(A) The modified fibroin with a reduced content of _n motif has 1 to 3 (A) _n motifs in which the domain sequence is at least from the N-terminal side to the C-terminal side compared to naturally occurring fibroin. Each may have an amino acid sequence corresponding to the deletion of one (A) _n motif.

(A) The modified fibroin in which the content of the _n motif is reduced, the domain sequence of the modified fibroin is at least two consecutive from the N-terminal side to the C-terminal side compared to the naturally derived fibroin (A) _n motif And an amino acid sequence corresponding to the deletion of one (A) _n motif repeated in this order.

(A) modified fibroin content of _n motifs has been reduced, the domain sequence, amino acids corresponding to at least the N-terminal side 2 every other towards the C-terminal side (A) _n motifs lacking It may have a sequence.

(A) A modified fibroin with a reduced content of _n- motif contains a domain sequence represented by Formula 1: [(A) _n- motif-REP] _m , and is adjacent to the C-terminal side from the N-terminal side. By sequentially comparing the number of amino acid residues in the REP of the two matching [(A) _n motif-REP] units, when the number of amino acid residues in the REP with a small number of amino acid residues is 1, the amino acid residue of the other REP The maximum total value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units with a cardinal ratio of 1.8 to 11.3 is x, and the total domain sequence 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, where y is the number of amino acid residues. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues).

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

Two adjacent [(A) _n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so as not to overlap. At this time, an unselected [(A) _n motif-REP] unit may exist. FIG. 1 includes 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 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison), pattern 4 (first REP and Comparison of the second REP). There are other selection methods.

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

In FIG. 1, when the smaller number of amino acid residues is 1, the ratio of the number of other amino acid residues is 1.8 to 11.3 [(A) _n motif-REP] unit pairs Shown in solid line. Hereinafter, such a ratio is referred to as a jagged ratio. When the smaller number of amino acid residues is 1, the ratio of the number of other amino acid residues is less than 1.8 or more than 11.3. [(A) _n motif-REP] unit pairs are indicated by broken lines. Indicated.

In each pattern, the number of all amino acid residues of two adjacent [(A) _n motif-REP] units indicated by solid lines is added (not only REP but also (A) the number of amino acid residues of the _n motif. is there.). Then, the total value added is compared, and the total value (maximum value of the total value) of the pattern having the maximum total value is set 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 of the domain sequence.

(A) In the modified fibroin in which the content of the _n motif is reduced, x / y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, It is still more preferably 70% or more, still more preferably 75% or more, and particularly preferably 80% or more. There is no restriction | limiting in particular in the upper limit of x / y, For example, you may be 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 / 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.

(A) A plurality of modified fibroin in which the content of _n motif is reduced is present in the domain sequence. (A) When at least 7 of _n motifs are modified fibroin composed only of alanine residues, x / y is 46.4% or more, preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, and 70% or more. Even more preferable, 80% or more is particularly preferable. There is no restriction | limiting in particular in the upper limit of x / y, and what is necessary is just 100% or less.

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

(A) As a more specific example of a modified fibroin with a reduced content of _n motif, (3-i) an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or ( 3-ii) A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 can be mentioned.

The modified fibroin (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 2 has the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin deleted from the N-terminal side to the C-terminal side every two (A) _n motifs Furthermore, one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting all GGX in REP of the amino acid sequence shown in SEQ ID NO: 2 with GQX. The amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.

The value of x / y of the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally-occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 4 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 12 is 89.3%. The z / w values in the amino acid sequences shown in SEQ ID NOs: 1, 2, 4, 10 and 12 are 46.8%, 56.2%, 70.1%, 66.1% and 70.0%, respectively. is there.

The modified fibroin (3-i) may be composed of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin (3-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and from the N-terminal side to the C-terminal side 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 residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio is 1: 1.8 to 11.3) It is preferable that x / y is 64.2% or more, where x is the maximum total value of the total number of bases and y is the total number of amino acid residues in the domain sequence.

The above-described modified fibroin may contain the tag sequence described above at one or both of the N-terminal and C-terminal.

More specific examples of the modified fibroin containing the tag sequence include (3-iii) an amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 7 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

(3-iii) The modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The modified fibroin (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and from the N-terminal side to the C-terminal side. 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 X is the maximum total value of the total number of amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. When the total number of amino acid residues of the domain sequence is y, x / y is preferably 64.2% or more.

The modified fibroin in which the content of glycine residue and (A) the content of _n motif is reduced, the domain sequence thereof is (A) the content of _n motif is reduced compared to naturally occurring fibroin. In addition, it has an amino acid sequence with a reduced content of glycine residues. In addition to the deletion of at least one or more (A) _n motifs, the domain sequence of the modified fibroin is different from that of naturally occurring fibroin in addition to at least one or more glycine residues in REP. It can be said to have an amino acid sequence corresponding to substitution with an amino acid residue. That is, it is a modified fibroin having the characteristics of the modified fibroin in which the content of the glycine residue is reduced and (A) the modified fibroin in which the content of the _n motif is reduced. Specific embodiments and the like are as described in the modified fibroin in which the content of glycine residues is reduced and (A) the modified fibroin in which the content of _n motif is reduced.

As a more specific example of a modified fibroin with reduced glycine residue content and (A) _n- motif content, (4-i) the amino acid represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the sequence (4-ii) SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 can be mentioned. Specific embodiments of the modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.

The modified fibroin according to another embodiment has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and It may have an amino acid sequence including a region having a large hydrophobic index locally, corresponding to the insertion of one or more amino acid residues having a large hydrophobic index in REP.

The region where the hydrophobic index is locally large is preferably composed of 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). More preferably, it is a residue.

The modified fibroin according to the present embodiment has one or more amino acid residues in REP substituted with amino acid residues having a large hydrophobicity index and / or 1 in REP compared to naturally occurring fibroin. Alternatively, in addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, one or more amino acid residues are substituted, deleted, inserted and / or compared with naturally occurring fibroin. Alternatively, there may be an amino acid sequence modification corresponding to the addition.

The modified fibroin according to the present embodiment, for example, hydrophobicizes one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in REP from the gene sequence of naturally-derived fibroin that has been cloned. It can be obtained by substituting amino acid residues (for example, amino acid residues having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in REP. In addition, for example, one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to insertion of, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more hydrophilic amino acid residues in 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 acids in REP In addition to the modification corresponding to the insertion of a 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.

Furthermore, the modified fibroin according to another embodiment includes a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and (A) located at the most C-terminal side of the domain sequence from the _n motif. The total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence obtained by removing the sequence up to the C-terminal from the domain sequence. P, and (A) _where the total number of amino acid residues contained in the sequence excluding the sequence from the _n motif to the C terminus of the domain sequence from the domain sequence is q / Q may have an amino acid sequence of 6.2% or more.

As for the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of bio.p. 7”. 105-132). Specifically, the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

The method for calculating p / q will be described in more detail. For the calculation, a sequence obtained by removing the sequence from the domain sequence represented by Formula 1: [(A) _n motif-REP] _m to the most C-terminal side from the domain (A) _n motif to the C terminus of the domain sequence. (Hereinafter referred to as “array A”). First, in all REPs included in the sequence A, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in the four consecutive amino acid residues by 4 (number of amino acid residues). The average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of hydrophobicity index is 2.6 or more”, 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. The total number of amino acid residues contained in sequence A is q.

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

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

The modified fibroin according to this embodiment includes, for example, one or a plurality of hydrophilic amino acid residues (for example, hydrophobicity) in the REP so that the amino acid sequence of the naturally-derived fibroin thus cloned satisfies the above p / q condition. Substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or one or more hydrophobic amino acid residues during REP Can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Alternatively, for example, an amino acid sequence satisfying the above p / q conditions can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis. In any case, compared to naturally occurring fibroin, one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index and / or one or more amino acid residues in REP. In addition to modifications corresponding to insertion of amino acid residues having a large hydrophobicity index, modifications corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. .

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

As another specific example of the modified fibroin, (5-i) the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23, or (5-ii) SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23 And a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by

The modified fibroin (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 19 is an amino acid sequence in which the alanine residues in the (A) _n motif of (A) naturally derived fibroin are deleted so that the number of consecutive alanine residues is five. . The amino acid sequence represented by SEQ ID NO: 20 is inserted into the amino acid sequence represented by SEQ ID NO: 19 by two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP, and represented by SEQ ID NO: 19. A part of amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence is almost the same. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two alanine residues at the C-terminal side of each (A) _n motif with respect to the amino acid sequence represented by SEQ ID NO: 19, and further adding some glutamine (Q) residues. A group is substituted with a serine (S) residue, and a part of amino acids on the C-terminal side is deleted so as to be approximately the same as the molecular weight of the amino acid sequence represented by SEQ ID NO: 19. The amino acid sequence represented by SEQ ID NO: 22 is obtained by inserting one amino acid sequence (VLI) consisting of 3 amino acid residues at every other REP to the amino acid sequence represented by SEQ ID NO: 21. The amino acid sequence shown in SEQ ID NO: 23 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues into the amino acid sequence shown in SEQ ID NO: 21 every other REP.

The modified fibroin (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23.

The modified fibroin (5-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23, and is located at the most C-terminal side (A) _n In all REPs included in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence, the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues, and (A) When the total number of amino acid residues contained in the sequence excluding the sequence from the _n motif to the C terminus of the domain sequence from the domain sequence is q , P / q is preferably 6.2% or more.

The above-mentioned modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal.

As more specific examples of modified fibroin comprising a tag sequence, (5-iii) the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24, SEQ ID NO: 25 or Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 26.

The amino acid sequences represented by SEQ ID NOs: 24, 25 and 26 are the amino acid sequences represented by SEQ ID NO: 5 (including His tag sequence and hinge sequence) at the N-terminus of the amino acid sequences represented by SEQ ID NOs: 20, 22 and 23, respectively. It is added.

(5-iii) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

The modified fibroin (5-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located at the most C-terminal side (A) _n In all REPs included in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence, the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues, and (A) When the total number of amino acid residues contained in the sequence excluding the sequence from the _n motif to the C terminus of the domain sequence from the domain sequence is q , P / q is preferably 6.2% or more.

The modified fibroin according to still another embodiment has an amino acid sequence in which the content of glutamine residues is reduced as compared with naturally occurring fibroin.

The modified fibroin according to this embodiment preferably includes at least one motif selected from a GGX motif and a GPGXX motif in the amino acid sequence of REP.

When the modified fibroin according to the present embodiment contains a GPGXX motif in REP, the content ratio of the GPGXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more. There is no restriction | limiting in particular in the upper limit of GPGXX motif content rate, 50% or less may be sufficient and 30% or less may be sufficient.

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 (modified fibroin or naturally derived) containing a domain sequence represented by the _n motif In fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the domain sequence (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence. The number obtained by multiplying the total number by three (ie, corresponding to the total number of G and P in the GPGXX motif) is s, and is located at the most C-terminal side. (A) The sequence from the _n motif to the C-terminal of the domain sequence is (A) The content ratio of the GPGXX motif is calculated as s / t, where t is the total number of amino acid residues of all REPs excluding the _n motif. It is.

In the calculation of the content ratio of the GPGXX motif, “A sequence located at the most C-terminal side (A) excluding the sequence from the _n motif to the C-terminal of the domain sequence from the domain sequence” (A) The sequence from the _n motif to the C terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence that is not highly correlated with the sequence characteristic of fibroin, and m is small In this case (that is, when the domain sequence is short), the calculation result of the content ratio of the GPGXX motif is affected, so this influence is excluded. When the “GPGXX motif” is located at the C-terminus of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.

FIG. 3 is a schematic diagram showing the domain sequence of the modified fibroin. The calculation method of the content ratio of GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 3 ("[(A) _n motif-REP] _m- (A) _n motif" type), all REPs are "most C-terminally located. (A) Since it is included in the “sequence excluding the sequence from the _n motif to the C-terminal of the domain sequence from the domain sequence” (the sequence indicated by “region A” in FIG. 3), The number of GPGXX motifs is 7, and s is 7 × 3 = 21. Similarly, all REPs are “a sequence located at the most C-terminal side (A) The sequence from the _n motif to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence indicated by “region A” in FIG. 1). )), The total number t of amino acid residues of all REPs excluding (A) the _n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t. In the case of the modified fibroin in FIG. 3, 21/150 = 14.0%.

The modified fibroin according to this embodiment preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and preferably 0%. Particularly preferred.

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 (modified fibroin or naturally derived) containing a domain sequence represented by the _n motif In fibroin), (A) the sequence from the _n- motif to the C-terminus of the domain sequence located on the most C-terminal side (sequence corresponding to “region A” in FIG. 3) excluding the sequence from the domain sequence. In the REP, the total number of glutamine residues contained in the region is u, the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded 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 rate, the reason why "A sequence located at the most C-terminal side (A) excluding the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

In the modified fibroin according to the present embodiment, the domain sequence has one or more glutamine residues in REP deleted or substituted with other amino acid residues as compared to naturally occurring fibroin. It may have a corresponding amino acid sequence.

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

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), 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, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. More preferred is an amino acid residue selected from among isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

In the modified fibroin according to the present embodiment, the hydrophobicity of REP is preferably −0.8 or more, more preferably −0.7 or more, still more preferably 0 or more, and It is still more preferable that it is 3 or more, and it is especially preferable that it is 0.4 or more. There is no restriction | limiting in particular in the upper limit of the hydrophobicity of REP, It may be 1.0 or less and may be 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 (modified fibroin or naturally derived) containing a domain sequence represented by the _n motif In fibroin), (A) the sequence from the _n- motif to the C-terminus of the domain sequence located on the most C-terminal side (sequence corresponding to “region A” in FIG. 1) is included. In the REP, the sum of the hydrophobicity index of each amino acid residue in the region is represented by v, and the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the _n motif. In the calculation of the hydrophobicity of REP, the reason why “A sequence located at the most C-terminal side (A) excluding the sequence from the _n motif to the C-terminal of the domain sequence from the domain sequence” is the reason described above. It is the same.

The modified fibroin according to the present embodiment has a domain sequence in which one or more glutamine residues in REP are deleted compared to naturally-occurring fibroin and / or one or more glutamine in REP. In addition to modifications corresponding to substitution of residues with other amino acid residues, there are further alterations in amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues. Also good.

The modified fibroin according to the present embodiment includes, for example, deletion of one or more glutamine residues in REP from the cloned gene sequence of natural fibroin and / or one or more glutamine residues in REP. Can be obtained by substituting with other amino acid residues. In addition, for example, one or more glutamine residues in REP are deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP are replaced with other amino acid residues. In particular, it can also be obtained by designing a corresponding amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As more specific examples of the modified fibroin according to the present invention, (6-i) the amino acid represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39 90% or more of the modified fibroin containing the sequence, or (6-ii) the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39 Mention may be made of modified fibroin comprising amino acid sequences having sequence identity.

The (6-i) modified fibroin will be described.

Amino acid sequence shown in SEQ ID NO: 4 (Met-PRT410) is a fibroin naturally occurring Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415) based on the nucleotide sequence and amino acid sequence of, _{(A) n} The amino acid sequence in which the alanine residue in the motif is continued is modified with an amino acid to improve productivity, such as the number of consecutive alanine residues is five. On the other hand, since Met-PRT410 has not altered the glutamine residue (Q), the glutamine residue content is comparable to the glutamine residue content of naturally occurring fibroin.

The amino acid sequence represented by SEQ ID NO: 27 (M_PRT888) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VL.

The amino acid sequence represented by SEQ ID NO: 28 (M_PRT965) is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with TS and substituting the remaining Q with A.

The amino acid sequence (M_PRT889) represented by SEQ ID NO: 29 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VL and replacing the remaining Q with I.

The amino acid sequence (M_PRT916) represented by SEQ ID NO: 30 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VI and replacing the remaining Q with L.

The amino acid sequence represented by SEQ ID NO: 31 (M_PRT918) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VF and replacing the remaining Q with I.

The amino acid sequence (M_PRT525) represented by SEQ ID NO: 37 is obtained by inserting two alanine residues into a region (A ₅ ) where alanine residues are continuous with respect to Met-PRT410 (SEQ ID NO: 4). The two C-terminal domain sequences were deleted and 13 glutamine residues (Q) were replaced with serine residues (S) or proline residues (P) so that they were almost the same as those in FIG.

The amino acid sequence represented by SEQ ID NO: 38 (M_PRT699) is obtained by substituting VL for all QQs in M_PRT525 (SEQ ID NO: 37).

The amino acid sequence represented by SEQ ID NO: 39 (M_PRT698) is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 37) with VL and replacing the remaining Q with I.

The amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38, and SEQ ID NO: 39 all have a glutamine residue content of 9% or less (Table 2). ).

The modified fibroin (6-i) may be composed of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 17. .

The modified fibroin of (6-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39. The amino acid sequence having The modified fibroin of (6-ii) is also represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. A protein containing a sequence. The sequence identity is preferably 95% or more.

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

As more specific examples of modified fibroin containing a tag sequence, (6-iii) amino acids represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 20 90% or more of the modified fibroin containing the sequence, or (6-iv) the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 20 Mention may be made of modified fibroin comprising amino acid sequences having sequence identity.

The amino acid sequences shown by SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 and SEQ ID NO: 41 are SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, respectively. The amino acid sequence represented by SEQ ID NO: 5 (including His tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 31, SEQ ID NO: 38 and SEQ ID NO: 39. Since only the tag sequence was added to the N-terminus, there was no change in glutamine residue content, and SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, and SEQ ID NO: 41. The glutamine residue content is 9% or less in any of the amino acid sequences indicated by (Table 3).

The modified fibroin of (6-iii) may be composed of the amino acid sequence represented by SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 or SEQ ID NO: 41. .

The modified fibroin (6-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 or SEQ ID NO: 41. The amino acid sequence having The modified fibroin of (6-iv) is also a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. A protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, using the gene encoding natural fibroin, the nucleic acid is produced by a method such as amplification by polymerase chain reaction (PCR), cloning, modification by genetic engineering techniques, or chemical synthesis. can do. The method for chemically synthesizing nucleic acids is not particularly limited. For example, AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) is used based on the amino acid sequence information of proteins obtained from the NCBI web database. A gene can be chemically synthesized by a method of linking oligonucleotides that are synthesized automatically by PCR or the like. At this time, in order to facilitate the purification and / or confirmation of the protein, a nucleic acid encoding a protein 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 terminus of the above amino acid sequence is synthesized. Also good.

Regulatory sequences are sequences that control the expression of modified fibroin in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, 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 expression of the modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value.

The type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like. As the expression vector, a vector which can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a protein can be transcribed is preferably used.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be 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. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

When a prokaryote is used as a host, examples of a vector for introducing a nucleic acid encoding a protein include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like.

Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of the 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 a vector into which a nucleic acid encoding a modified fibroin is introduced include YEp13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method, and the like.

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

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 protein in the culture medium, and collecting the protein from the culture medium. The method for culturing a host in a culture medium can be performed according to a method usually used for culturing a 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, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.

Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism. Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc. Organic acids and alcohols such as ethanol and propanol can be used. Examples of the nitrogen source include ammonium salts of inorganic acids 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, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used. As inorganic salts, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

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

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

Isolation and purification of the expressed protein can be performed by a commonly used method. For example, when the protein is expressed in a dissolved state in the cell, the host cell is recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a Manton Gaurin. The host cells are disrupted with a homogenizer, dynomill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for protein isolation and purification, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent, etc. Precipitation method, anion exchange chromatography method using resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using resin such as S-Sepharose FF (manufactured by Pharmacia) Electrophoresis methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Using methods such as these alone or in combination, purification It is possible to obtain the goods.

In addition, when the protein is expressed by forming an insoluble substance in the cell, the host cell is similarly collected and then crushed and centrifuged to collect the protein insoluble substance as a precipitate fraction. The recovered protein insoluble matter can be solubilized with a protein denaturant. After the operation, a purified protein preparation can be obtained by the same isolation and purification method as described above. When the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture with a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.

<Method for producing modified fibroin fiber>
The modified fibroin fiber can be produced by a known spinning method. That is, for example, when producing a modified fibroin fiber containing a modified fibroin as a main component, first, the modified fibroin produced according to the above-described method is converted into dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), A dope solution is prepared by adding to a solvent such as formic acid or hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator, if necessary, and dissolving. Next, the modified fibroin fiber can be obtained by spinning using this dope solution by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning. Preferred spinning methods include wet spinning or dry wet spinning.

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

A spinning method using the spinning device 10 will be described. First, the dope solution 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8. In the lab scale, the dope solution may be filled into a cylinder and extruded from a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied into the coagulating liquid 11 in the coagulating liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulated body is formed. Next, the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14. Thereafter, the stretched fibrous solidified body is supplied to the drying device 4 and dried in the yarn path 22, and the modified fibroin fiber 36 is obtained as the wound body 5. Reference numerals 18a to 18g denote thread guides.

The coagulation liquid 11 may be any solvent that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulation liquid 11 may appropriately contain water. The temperature of the coagulation liquid 11 is preferably 0 to 30 ° C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the base 9, the extrusion speed is preferably 0.2 to 6.0 mL / hour per hole, and 1.4 to 4.0 mL / hour More preferably it is time. The distance through which the coagulated modified fibroin passes through the coagulating liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) may be long enough to efficiently remove the solvent. 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min. The residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11. The coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as necessary.

In addition, as the stretching performed when obtaining the modified fibroin fiber, for example, dry stretching is employed in addition to the above-described pre-stretching in the coagulating liquid tank 20 and the wet heat stretching performed in the stretching bath 21.

Wet heat stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating. The temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C. In wet heat drawing, undrawn yarn (or predrawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.

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

Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching. For example, wet heat stretching and dry heat stretching can be appropriately combined.

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

<Modified fibroin fiber>
The modified fibroin fiber according to the present embodiment may be one obtained by spinning the above-described modified fibroin, and in this case, includes the above-described modified fibroin as a main component.

The modified fibroin fiber has a shrinkage ratio A of, for example, 2% or more when it is brought into a wet state by contacting with water, and a shrinkage ratio B of 7% or more when it is dried.
Here, the shrinkage rate A is defined by the following equation.
Shrinkage ratio A = {1− (length of artificial fibroin fiber wetted by contact with water / length of artificial fibroin fiber before spinning and before contact with water)} × 100 (%)
On the other hand, the shrinkage rate B is defined by the following equation.
Shrinkage rate B = {1− (length of protein fiber that has undergone a shrinking step including contact with water having a boiling point / length of protein fiber before undergoing the shrinking step)} × 100 (%)

The modified fibroin fiber according to the present embodiment has a shrinkage rate A of 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more. Or 6% or more. The upper limit of the shrinkage rate A is not particularly limited, but 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% It may be the following. On the other hand, the shrinkage rate B is preferably more than 7%, more preferably 15% or more, further preferably 25% or more, still more preferably 32% or more, and 40% or more. Even more preferably, it is particularly preferably 48% or more, particularly preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more. preferable. The upper limit of the shrinkage rate A is usually 80% or less.

<Protein fiber yarn>
The protein fiber yarn according to the present embodiment is a single yarn that is one of a twisted yarn containing a modified fibroin fiber, a false twisted yarn containing a modified fibroin fiber, and a spun yarn containing a modified fibroin fiber. The protein fiber yarn, which is a raw material yarn used for knitting a knitted fabric or weaving a woven fabric, is mainly composed of modified fibroin fibers. This protein fiber yarn may consist of modified fibroin fibers. The protein fiber yarn contains modified fibroin as a main component. The raw material yarn used for knitting the knitted fabric may partially contain other fibers.

The raw material yarn may be a filament (long fiber) twisted yarn or false twisted yarn. The raw material yarn may be a spun yarn made of staples (short fibers). In the present embodiment, the raw material yarn used for knitting the knitted fabric is a single yarn. By using a single yarn, a texture that cannot be obtained with a double yarn can be realized in a knitted fabric.

The twist direction of the twisted yarn, false twisted yarn or spun yarn may be S twist (right twist) or Z twist (left twist).

In the present specification, the occurrence of skew in the knitted fabric or woven fabric or that is sufficiently suppressed is referred to as "no skew", such a knitted fabric is referred to as "no skew knitted fabric", Such a woven fabric is referred to as “oblique fabric”. Further, the distortion of the fabric refers to a defective state similar to the skew in the fabric. Presence / absence of skew and distortion can be determined by visual inspection of the stitch or the weave.

The fineness of the twisted yarn, false twisted yarn or spun yarn is preferably a suitable count of the gauge or loom of the knitting machine to be used, and spinning is performed accordingly.

A single yarn having a count of 13 to 150 Nm is thinner than a double yarn and has an excellent texture and softness. The yarn count of the single yarn is not particularly limited, and may be any count obtained by a known spinning method. The number of yarn twists is not particularly limited, and may be any number of twists used in well-known spinning.

<Manufacturing method of single yarn>
The method for producing a single yarn when the single yarn is a twisted yarn includes a twisting step of obtaining a twisted yarn (single yarn) by twisting a plurality of fibers including the modified fibroin fiber. This manufacturing method may include a heating step of heating the yarn in a state where the twist of the fiber is maintained. The twisting step and the heating step may be independent of each other or may be performed simultaneously.

In the twisting process, the modified fibroin fiber is twisted. As a means for adding twist, a known means can be used, for example, a twisting machine or the like can be used. As a twisting machine, a twisting machine corresponding to various known twisting types (up type, down type, special beam type, air beam type, rewirer, etc.), for example, Italy type twisting machine, double twister, ring twisting machine, composite A twisting machine, an interlace, a pirn winder, a mule spinning machine, etc. are mentioned. In the twisting step, the number of twists of the yarn is adjusted so as to be in the preferable range described above. The number of twists can usually be adjusted by changing the setting value of the twisting machine.

In addition, in the method for producing a single yarn when the single yarn is a twisted yarn, the twisted yarn is treated at a high humidity in a step including a twisting step, that is, a step up to the twisting step and a step including a winding step after the twisting step. You may implement the water addition and maintenance process which raises the moisture content in the fiber of a twisted yarn by exposing to an environment (1st humidity environment). In this case, the high humidity environment may be, for example, a humidity of 60% or more, or may be a humidity of 60% to 80%. The humidity is preferably 65% to 75%. The time for which the twisted yarn is exposed to the high humidity environment is not particularly limited, but may be 1 to 48 hours or 12 to 24 hours.

When the single yarn is a false twisted yarn, the method for producing the single yarn includes a twisting step of obtaining a twisted yarn (single yarn) by twisting a plurality of fibers including the modified fibroin fiber, and untwisting the twisted twisted yarn. An untwisting step of obtaining a false twisted yarn. This manufacturing method may include a heating step of heating the yarn while maintaining the twist of the fiber. The twisting step and the heating step may be independent of each other, may be performed simultaneously, and are preferably performed simultaneously.

The conditions described in the method for producing twisted yarn can be applied to the parts common to the method for producing false twisted yarn and the method for producing twisted yarn described above. Here, description is abbreviate | omitted and a different part from 1st embodiment is demonstrated.

In the twisting process, the modified fibroin fiber is twisted. As a means for adding twist, a known means can be used, for example, a false twisting machine or the like can be used. Examples of the false twisting machine include a pin false twisting machine, a friction false twisting machine, and a belt false twisting machine. In the untwisting step, the yarn that has undergone the twisting step is twisted in the opposite direction to the twisting step. The number of twists in the untwisting process may be equal to the number of twists in the twisting process, or may be equal to or greater than the number of twists in the twisting process. The number of twists in the untwisting step can be adjusted by changing the set value of the false twisting machine.

In the single yarn production method when the single yarn is a false twisted yarn, the twisted yarn or false twisted yarn is placed in a high-humidity environment (first step) in the step including the twisting step or the step including the twisting step and the untwisting step. A moisture addition / maintenance step for increasing the moisture content in the fiber of the twisted yarn or false twisted yarn may be performed by exposure to a humidity environment. In this case, the high humidity environment may be, for example, a humidity of 60% or more, or may be a humidity of 60% to 80%. The humidity is preferably 65% to 75%. The time for which the twisted yarn is exposed to the high humidity environment is not particularly limited, but may be 1 to 48 hours or 12 to 24 hours.

A method for producing a single yarn when the single yarn is a spun yarn includes a cutting step of cutting a filament (long fiber) containing modified fibroin fibers, and a spinning step of spinning a staple (short fiber) obtained in the cutting step, Is provided. Prior to the cutting step, this manufacturing method may include a crimping step in which the filament and moisture are brought into contact with each other, or a crimping step in which the staple and moisture are brought into contact with each other after the cutting step. May be. These crimping steps may include a drying step of drying the filaments or staples after contact of moisture with the filaments or staples. According to the crimping process, the texture of the spun yarn is further improved.

As a method (step) for crimping the filament, in addition to the above-described method (step) for crimping (wet crimping), the filament made of the modified fibroin is brought into contact with water (immersion or exposure) to be shrunk. A method of mechanically crimping filaments can also be used. As this mechanical crimping method, for example, a known method such as mechanical pressing or injection pressing can be employed. Both wet crimping and mechanical crimping may be performed, but only wet crimping or only mechanical crimping may be performed. In the case where both wet crimping and mechanical crimping are performed, the order of implementation is not limited. Although wet crimping may be performed first or mechanical crimping may be performed first, it is preferable to perform mechanical crimping first from the viewpoint of workability and the like. In particular, by performing wet crimping, skew in the knitted fabric that is a knitted product can be suppressed.

Water may be used as water in wet crimping, or steam may be used. The wet crimping may be performed in a batch manner or a continuous manner. When the filament is immersed in water, a suitable temperature range of water is, for example, 10 to 60 ° C. By setting the water temperature to 10 ° C. or higher, the time required for shrinkage can be shortened, and by setting the water temperature to 60 ° C. or lower, a decrease in fiber strength can be prevented. The amount of crimp (shrinkage rate) can be controlled by adjusting the water temperature. As the water temperature is higher in the range of 10 to 60 ° C., the amount of crimp (shrinkage rate) tends to increase. The immersion time in water is, for example, preferably 30 seconds or longer, more preferably 11 minutes or longer, or 1 to 10 minutes. By setting the immersion time to 1 minute or longer, a sufficient crimping effect can be obtained, and by setting the immersion time to 10 minutes or less, a decrease in productivity can be prevented. Since water is easy to handle, wet crimping is easy.

When steam is brought into contact with the filament, a suitable temperature range of the steam may be 100 to 230 ° C. or 100 to 120 ° C. By setting the steam temperature to 230 ° C. or lower, protein degradation can be prevented. When steam is used, a large shrinkage can be obtained in a short time.

The moisture in wet crimping may contain a volatile solvent or an oil dispersion. With water and a volatile solvent, the drying rate can be improved. According to water and the oil dispersion, wet crimping and oil adhesion can be performed simultaneously.

As an example of wet crimp using water, for example, after being immersed in hot water while feeding the filament from the bobbin, the filament is dried with hot air or a hot roller, and then the filament is continuously cut as necessary. A series of processes such as. On the other hand, any known crimping machine can be used for the mechanical crimping.

As the spinning process, a known method can be adopted. For example, when the spun yarn is made of a modified fibroin fiber, the spinning process may be a spun spinning process for spinning a protein crimped staple made of the modified fibroin fiber. This spinning process includes, for example, a blending process in which protein-crimped staples are uniformly mixed and a spinning solvent is added, and a fiber is split and formed into a sheet shape and laminated, and further finely divided and kneaded to form a bundle of yarns. Includes a card process to create, a mule process to create a desired yarn with a high degree of uniformity by stretching and twisting a bundle of yarns, and a winding process to wind up the spun yarn (single yarn) obtained in the mule process But you can. The mule process corresponds to a twisting process in which a single yarn is obtained by twisting fibers containing modified fibroin fibers. In the winding process, a known winder can be used. The winding process is a process of so-called cone-up, in which a spun yarn is wound into a cylindrical cheese or conical corn state.

Further, when the spun yarn is a mixed spinning made of a modified fibroin fiber and another fiber (heterogeneous fiber), the spinning process is a cotton spinning spinning process in which the protein crimped staple made of the modified fibroin fiber is mixed with another fiber. It may be. This spinning process consists of a fiber opening process for creating a wrap, a card process for creating a sliver by stretching the fibers in parallel, and a kneading process to make the fibers more parallel while combining several slivers to make the thickness uniform. A spinning process that stretches and thins the sliver and twists to create a roving yarn, a spinning process that stretches and twists the roving to create a yarn of a predetermined thickness, and a spinning process. A winding step of winding the obtained single yarn. The spinning process corresponds to a twisting process in which a single yarn is obtained by twisting fibers containing modified fibroin fibers. In the winding process, a known winder can be used. The winding process is a process of so-called cone-up, in which a spun yarn is wound into a cylindrical cheese or conical corn state.

In addition, in this specification, the other fiber means a fiber that does not contain modified fibroin, and examples thereof include chemical fiber and natural fiber. When used in combination with other fibers, the modified fibroin content is preferably 5% or more by mass, more preferably 20% or more by mass, based on the total amount of fibers including the modified fibroin fibers. The mass is more preferably 50% or more.

In the method for producing a single yarn when the single yarn is a spun yarn, at least in the twisting process, that is, the process up to the twisting process and the process including the winding process after the twisting process, You may implement the water addition and maintenance process which raises the moisture content in the fiber of a spun yarn by exposing to an environment (1st humidity environment). In this case, the high humidity environment may be, for example, a humidity of 60% or more, or may be a humidity of 60% to 80%. The humidity is preferably 65% to 75%. The time for which the twisted yarn is exposed to the high humidity environment is not particularly limited, but may be 1 to 48 hours or 12 to 24 hours.

In the moisture addition / maintenance process, the moisture content in the fiber is maintained so as not to decrease. The moisture addition / maintenance process includes a twisting process, a process preceding the twisting process (for example, a card process), and a process subsequent to the twisting process (for example, a mule process, a fine spinning process, a roving process, etc.) May also be implemented. That is, the moisture addition / maintenance process may be performed in substantially the entire spinning process, or may be performed in a part of the spinning process (including the twisting process).

For example, when producing spun yarn, spinning crimped staples that have been shrunk to prevent static electricity, etc., and spinning up to cone up (to be used for knitting) It may be carried out in the environment (humidity 65 to 75%).

Regardless of whether the single yarn is a twisted yarn, a false twisted yarn or a spun yarn, the method for producing the single yarn is after the twisting step (after twisting) and knitting the knitted fabric It is carried out before the step or the weaving step of the fabric, and includes a moisture content lowering step for reducing the moisture content of the single yarn. In other words, the moisture content lowering step is a drying step of drying the single yarn. The moisture content lowering step is a step of reducing the moisture content of the single yarn so that the moisture content of the single yarn is close to the moisture content of the protein fiber before being twisted. Here, the protein fiber before being twisted is a modified fibroin fiber that has been spun by the spinning device 10 (via the drying device 4). The moisture content of a single yarn is converted to the official moisture content.

Hereinafter, the moisture content lowering process will be described by taking as an example the case where the single yarn is a spun yarn. In the moisture content lowering step, the moisture content in the fiber of the spun yarn is lowered by exposing the spun yarn to a low humidity environment (second humidity environment). The moisture content lowering step is a step of storing, for example, a corn-up spun yarn in a room controlled at a predetermined humidity. The low humidity environment has a lower humidity than the high humidity environment, for example, a humidity of 50% or less. The low humidity environment is preferably a humidity of 30% or less, and more preferably a humidity of 20% or less. The time for which the spun yarn is exposed to a low humidity environment is not particularly limited, but may be 1 to 24 hours.

The moisture content of the spun yarn that has undergone the moisture content lowering step is, for example, 15% or less. The moisture content of the spun yarn that has undergone the moisture content lowering step may be 10% or less. The reduction rate of the moisture content by the moisture content reduction process is, for example, 2 to 10%.

In the moisture content lowering step, the spun yarn is dried to reduce the moisture content before cone use and before use in knitting. At this time, it is preferable to reduce the moisture content (moisture content) of the spun yarn to the same level as the moisture content of the crimped staple before the spinning step.

In the structural protein fiber yarn of this embodiment, the final effect can be obtained by changing the moisture content in the fiber during spinning and the moisture content in the fiber after cone-up. The spinning method may be any known method. The moisture change rate in the fiber that can achieve the final effect can be obtained by lowering the moisture content in the cone-up state by controlling the moisture content during the process from spinning to cone-up and the moisture content after the cone-up. The final effect can be obtained from 1 to 24 hours after the moisture content is lowered in the corn-up state (after completion of the moisture content reduction step).

Note that the method of reducing the moisture content is not limited to the above. The moisture content lowering step may be any of drying by heat, drying by lowering the atmospheric pressure, natural drying, and the like. Further, as a method for reducing the moisture content, the spun yarn may be immersed in an organic solvent.

<Method for producing woven fabric and knitted fabric>
Knitting or weaving is performed using the twisted yarn or false twisted yarn obtained from the filament as described above, or the spun yarn obtained from the crimped filament or crimped staple. A known loom or knitting machine can be used.

Knitted fabric can be obtained by a known knitting method. As the knitting machine used, it is more preferable to use a non-sewn knitting machine because a raw material knitted fabric can be manufactured in the form of a final product, particularly with all known looms and known knitting machines.

According to the method for producing a protein fiber yarn and the method for producing a woven fabric of the present embodiment described above, for example, in a woven fabric using a spun yarn having a reduced moisture content (moisture content), skew and distortion Occurrence is suppressed. On the other hand, when knitting is performed using spun yarn with a high moisture content without drying, skewing and distortion occur in the finished woven body. In the spun yarn of the present embodiment, the residual torque is reduced due to a decrease in moisture content, thereby suppressing skewing, but at the same time, the amount and length of fluff can be reduced. As a result, the occurrence of pilling can be suppressed.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples.

<Manufacture of modified spider silk fibroin>
(1) Preparation of plasmid expression strain Based on the base sequence and amino acid sequence of fibroin (GenBank accession numbers: P46804.1, GI: 1174415) derived from Nephila clavipes, the amino acid sequence represented by SEQ ID NO: 13 A modified fibroin (hereinafter also referred to as “PRT799”) was designed. The amino acid sequence represented by SEQ ID NO: 13 has an amino acid sequence in which substitution, insertion, and deletion of amino acid residues are performed for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes. Furthermore, an amino acid sequence (tag sequence and hinge sequence) represented by SEQ ID NO: 5 is added to the N-terminus.

Next, a nucleic acid encoding PRT799 was synthesized. The nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence represented by SEQ ID NO: 13. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium (Table 4) containing ampicillin so that the OD ₆₀₀ was 0.005. The culture temperature was kept at 30 ° C., and flask culture was performed until the OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

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

Immediately after the glucose in the production medium was completely consumed, a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and cultured for 20 hours. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein. At the time when 20 hours passed after the addition of IPTG, the culture solution was centrifuged, and the cells were collected. SDS-PAGE is performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of the target spider silk fibroin by the appearance of the target spider silk fibroin size band depending on the addition of IPTG. It was confirmed.

(3) Protein purification The cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 30 ° C. at 30 ° C. Stir with a stirrer for 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 aggregated protein obtained after dialysis was collected by centrifugation, water was removed with a freeze dryer, and the lyophilized powder was collected to obtain a modified spider silk fibroin “PRT799”.

<Preparation of modified spider silk fibroin fiber>
(1) Preparation of dope solution After adding the above modified spider silk fibroin (PRT799) to dimethylsulfoxide (DMSO) to a concentration of 24% by mass, LiCl as a dissolution accelerator is adjusted to a concentration of 4.0% by mass. Added to. Thereafter, using a shaker, the modified spider silk fibroin was dissolved over 3 hours to obtain a DMSO solution. Dust and bubbles in the obtained DMSO solution were removed to prepare a dope solution. The solution viscosity of the dope solution was 5000 cP (centipoise) at 90 ° C.

(2) Spinning Known dry and wet spinning was performed using the dope solution obtained as described above and the spinning device 10 shown in FIG. 4 to obtain a filament made of modified spider fiber fibroin. Here, dry and wet spinning was performed under the following conditions.
Coagulation liquid (methanol) temperature: 5-10 ° C
Stretch ratio: 4.52 times Drying temperature: 80 ° C
Then, this filament was wound around a bobbin.

<Example 1>
A plurality of modified spider silk filaments (artificial spider silk filaments) obtained as described above and wound on a bobbin are bundled and cut into a length of 38 mm with a table-type fiber cutter to produce an artificial spider silk staple did. The produced artificial spider silk staple was crimped by being immersed in water at 40 ° C. for 1 minute to be crimped, and then dried at 40 ° C. for 18 hours to obtain a crimped staple. The crimped staple was sprayed with 4% water and 2% oil as a spinning oil for the same mass. The spinning oil was infiltrated for 12 hours in a hermetically sealed state.

After spinning with a four-spinning spinning machine and then stretching with a mule spinning machine, spinning was performed so that the draft rate was 1.45, the twist number was Z340, and the target spinning number was 30 Nm. Each part of the machine was adjusted. During this process, the resulting cup was connected and cone-up was performed. The humidity in the factory was adjusted to 75%, and the moisture content in the fiber was controlled so as not to decrease. The fiber moisture content during this process was 14-12% (in terms of official moisture content). Thereafter, it was stored in a corn-up state for 24 hours and in a cabinet at room temperature of 20%. The fiber moisture content after 24 hours was 9% (in terms of official moisture content). Using the finished spun yarn, a tengu structure was knitted with a 14G circular knitting machine, and it was confirmed that skewing occurred. The obtained knitted fabric is shown in FIG.

As shown in FIG. 5, a knitted fabric was produced by knitting a single yarn of spun yarn made of artificial spider yarn fibroin fiber (for reference, count: 30 Nm, twist: 340 T / m) with a flat knitting machine, No skew was seen, and a non-skew knitted fabric could be obtained. The single yarn having a count of 30 Nm was thin, and the knitted fabric as the knitted product exhibited excellent texture and softness.

<Comparative Example 1>
Artificial spider silk staples were produced in the same manner as in Example 1 to obtain crimped staples. The crimped staple was spun using a known spinning equipment to obtain a spun yarn having a fineness of 18 Nm and a twist number of 340 T / m, and cone-up. After corn-up, the knitted fabric is knitted by a tense knitting machine with a flat knitting machine without performing a special operation to reduce the moisture content in the spun yarn (that is, after 1 hour without storing for a long time). did. The obtained knitted fabric is shown in FIG.

As shown in FIG. 6, when a knitted fabric was produced by knitting a single yarn of spun yarn of artificial spider yarn fibroin fiber having a count of 18 Nm and a twist number of 340 T / m with a flat knitting machine, skew was observed. It was.

According to the present invention, for example, the moisture content after the cone-up of the protein fiber yarn is lowered than the moisture content in the process before the cone-up including at least the twisting process, thereby causing skewing and distortion. The obtained residual torque can be eliminated. Furthermore, since the protein fiber yarn according to the present invention has a small residual torque, it is difficult for fluff to occur. Therefore, the occurrence of pilling can be suppressed.

DESCRIPTION OF SYMBOLS 1 ... Extruding device, 2 ... Undrawn yarn manufacturing device, 3 ... Wet heat drawing device, 4 ... Drying device, 6 ... Dope solution, 10 ... Spinning device, 20 ... Coagulating liquid tank, 21 ... Drawing bath, 36 ... Modified spider yarn Fibroin fiber.

Claims

A single yarn that is one of a twisted yarn containing modified fibroin fiber, a false twisted yarn containing modified fibroin fiber, and a spun yarn containing modified fibroin fiber, and its moisture content can be reduced after twisting. Protein fiber yarn.
A single yarn of the spun yarn containing a modified fibroin fiber, which is exposed to a first humidity environment in a twisting process of twisting at least the modified fibroin fiber, and after the twisting process, from the first humidity environment The protein fiber yarn according to claim 1, wherein the moisture content is lowered by exposure to a second humidity environment having a low humidity.
The protein fiber yarn according to claim 1 or 2, wherein the modified fibroin is a modified spider silk fibroin.
A knitted fabric obtained by knitting or weaving the protein fiber yarn according to any one of claims 1 to 3.
A method for producing a protein fiber yarn that produces a single yarn that is one of a twisted yarn containing a modified fibroin fiber, a false twisted yarn containing a modified fibroin fiber, and a spun yarn containing the modified fibroin fiber,
A twisting step of obtaining the single yarn by twisting a fiber containing the modified fibroin fiber;
A method for producing a protein fiber yarn, comprising: a moisture content reduction step that is performed after the twisting step and reduces the moisture content of the single yarn.
A spinning step of spinning a single yarn of a spun yarn containing the modified fibroin fiber, the spinning step including the twisting step of twisting the modified fibroin fiber in a first humidity environment;
The moisture content lowering step, which is performed after the spinning step and lowers the moisture content of the single yarn under a second humidity environment having a humidity lower than the first humidity environment. The manufacturing method of the protein fiber yarn of description.
The humidity under the first humidity environment is 60% or more, the humidity under the second humidity environment is 50% or less, and the moisture content of the single yarn is 15% or less (official) in the moisture content lowering step The method for producing a protein fiber yarn according to claim 6, wherein the protein fiber yarn is reduced to a moisture content conversion).
The method for producing a protein fiber yarn according to any one of claims 5 to 7, wherein the modified fibroin is a modified spider silk fibroin.
A method for producing a knitted fabric, wherein the single yarn produced by the method for producing a protein fiber yarn according to any one of claims 5 to 8 is knitted or woven.