EP3800286A1

EP3800286A1 - High-shrinkage artificial fibroin spun yarn, method for manufacturing same, artificial fibroin spun yarn, and method for shrinking same

Info

Publication number: EP3800286A1
Application number: EP19782354.5A
Authority: EP
Inventors: Takumi IGARASHI; Atsushi Ikeda; Yunosuke ABE; Seiji Harada
Original assignee: Kojima Industries Corp; Spiber Inc
Current assignee: Kojima Industries Corp; Spiber Inc
Priority date: 2018-04-03
Filing date: 2019-04-03
Publication date: 2021-04-07
Also published as: EP3800286A4; JPWO2019194245A1; JP7340262B2; CN112534090A; CN112534090B; WO2019194245A1

Abstract

The present invention relates to a high-shrinkage artificial fibroin spun yarn which is a shrunk artificial fibroin spun yarn including a modified fibroin and having a shrinkage rate of greater than 1% defined by the following Formula I: Shrinkagerate=1−lengthofshrunkartificialfibroinspunyarn/lengthofartificialfibroinspunyarnafterspinningandbeforecontactwithwater×100%

Description

Technical Field

The present invention relates to a high-shrinkage artificial fibroin spun yarn and a method for manufacturing the same, and an artificial fibroin spun yarn and a method for shrinking the same.

Background Art

In the related art, spun yarns made of synthetic fibers, natural fibers, or the like have been used for various knitted fabric materials. Thus, when knitted fabrics are manufactured, spun yarns that can satisfy requirements for the knitted fabrics are selected. For example, when clothes, bedclothes, and the like having an expensive feeling and being luxurious are manufactured, silk spun yarns and the like are selected as materials thereof.
In addition, depending on the properties required for knitted fabrics, spun yarns as materials therefor may be subjected to predetermined processing. For example, in clothes, bedclothes and the like, when higher flexibility and heat retention are desired in addition to an expensive feeling and a luxurious feeling, silk spun yams that have been subjected to shrinkage processing such that bulkiness may be increased may be used.
Regarding a silk shrinkage method, for example, so-called salt shrinkage processing in which silk is immersed in an aqueous solution in which inorganic salts such as calcium nitrate and calcium chloride are dissolved in a high concentration (salt shrinkage solution) and shrunk is known (for example, Patent Literature 1).
On the other hand, for example, synthetic fibers such as polyester fibers, polyamide fibers and acrylic fibers which are generally used for clothes, bedclothes, and the like may be brought into contact with boiling water, and thus a shrinkage rate of 40% or more may be realized (Patent Literature 2).

Citation List

Patent Literature

[Patent Literature 1] Japanese Unexamined Patent Publication No. 2001-64866
[Patent Literature 2] Japanese Unexamined Patent Publication No. 2009-121003

Summary of Invention

Technical Problem

However, silk has a very small shrinkage rate when it is simply brought into contact with water. In addition, the shrinkage method described in Patent Literature 2 has a high risk because boiling water with a high temperature is handled.
An objective of the present invention is to provide a high-shrinkage artificial fibroin spun yarn which has a sufficiently high shrinkage rate and an excellent tactile feel and flexibility, and also can be manufactured safely and a method for manufacturing the same. Another objective of the present invention is to provide an artificial fibroin spun yarn which allows a high-shrinkage artificial fibroin spun yarn to be manufactured at a sufficiently high shrinkage rate and safely, and a method for shrinking the same.

Solution to Problem

For example, the present invention relates to the following inventions.

[1] A high-shrinkage artificial fibroin spun yarn which is a shrunk artificial fibroin spun yarn including a modified fibroin and having a shrinkage rate of greater than 1% defined by the following Formula I: $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$
[2] The high-shrinkage artificial fibroin spun yarn according to [1],
wherein the modified fibroin is a modified spider silk fibroin.
[3] The high-shrinkage artificial fibroin spun yarn according to [1] or [2], which is shrunk when brought into contact with water having a temperature lower than a boiling point.
[4] The high-shrinkage artificial fibroin spun yarn according to [3],
wherein the temperature of the water is 10 to 90°C.
[5] The high-shrinkage artificial fibroin spun yarn according to [3] or [4], which is additionally shrunk when drying is performed after contact with the water.
[6] A method for manufacturing a high-shrinkage artificial fibroin spun yarn, including:
- a shrinkage process in which an artificial fibroin spun yarn including a modified fibroin is brought into contact with water having a temperature lower than a boiling point,
- wherein a shrinkage rate defined by the following Formula I is greater than 1%: $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$
[7] The method for manufacturing a high-shrinkage artificial fibroin spun yarn according to [6],
wherein the modified fibroin is a modified spider silk fibroin.
[8] The method for manufacturing a high-shrinkage artificial fibroin spun yarn according to [6] or [7],
wherein the temperature of the water is 10 to 90°C.
[9] The method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [8],
wherein the shrinkage process further includes drying of the artificial fibroin spun yarn after it is brought into contact with the water.
[10] A method for shrinking an artificial fibroin spun yarn, including:
- a shrinkage process in which an artificial fibroin spun yarn including a modified fibroin is brought into contact with water having a temperature lower than a boiling point,
- wherein a shrinkage rate defined by the following Formula I is greater than 1%: $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$
[11] The method for shrinking an artificial fibroin spun yarn according to [10], wherein the modified fibroin is a modified spider silk fibroin.
[12] The method for shrinking an artificial fibroin spun yarn according to [10] or [11],
wherein the temperature of the water is 10 to 90°C.
[13] The method for shrinking an artificial fibroin spun yarn according to any one of [10] to [12],
wherein the shrinkage process further includes drying of the artificial fibroin spun yarn after it is brought into contact with the water.
[14] An artificial fibroin spun yarn including a modified fibroin and having a shrinkage rate of greater than 1% defined by the following Formula II: $Shrinkage rate = \{1 - (length of artificial fibroin spun yarn subjected to shrinkage processing including being brought into contact with water having a temperature lower than a boiling point / length of artificial fibroin spun yarn before shrinkage processing is performed)\} \times 100 (%)$
[15] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein the domain sequence includes an amino acid sequence with a reduced content of the (A)_n motif, which corresponds to deletion of at least one or more (A)_n motifs, as compared with a naturally derived fibroin:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
[16] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [15], wherein the domain sequence includes an amino acid sequence corresponding to a case in which at least one (A)_n motif for every one to three (A)_n motifs from the N-terminal side to the C-terminal side is deleted, as compared with a naturally derived fibroin.
[17] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [15], wherein the domain sequence includes an amino acid sequence corresponding to a case in which, from the N-terminal side to the C-terminal side, at least deletion of two consecutive (A)_n motifs and deletion of one (A)_n motif are repeated in that order, as compared with a naturally derived fibroin.
[18] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein, when the numbers of amino acid residues of REP of adjacent two [(A)_n motif-REP] units are sequentially compared from the N-terminal side to the C-terminal side, the maximum value of the total value obtained by adding the number of amino acid residues of the adjacent two [(A)_n motif-REP] units in which a ratio of the number of amino acid residues of the other REP with respect to 1 set for the number of amino acid residues of one REP with a smaller number of amino acid residues is 1.8 to 11.3 is set as x, and the total number of amino acid residues of the domain sequence is set as y, x/y is 50% or more:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
[19] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein the domain sequence includes an amino acid sequence with a reduced content of glycine residues, which corresponds to substitution of at least one or more glycine residues in REP with other amino acid residues, as compared with a naturally derived fibroin:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
[20] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [19],
wherein the domain sequence includes an amino acid sequence corresponding to a case in which, in at least one motif sequence selected from among GGX and GPGXX (where, X represents an amino acid residue other than glycine) in REP, one glycine residue in at least one or more motif sequences is substituted with another amino acid residue, as compared with a naturally derived fibroin.
[21] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [20],
wherein a proportion of the motif sequences in which a glycine residue is substituted with another amino acid residue with respect to all motif sequences is 10% or more.
[22] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein, when the total number of amino acid residues of amino acid sequences composed of XGX (where, X represents an amino acid residue other than glycine) contained in all REPs in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as z, and the total number of amino acid residues in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as w, z/w is 50.9% or more:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
[23] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [19] to [22],
wherein the modified fibroin includes an amino acid sequence corresponding to a case in which one or more glycine residues in REP are substituted with other amino acid residues and additionally, one or more amino acid residues are substituted, deleted, inserted and/or added, as compared with a naturally derived fibroin.
[24] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein the domain sequence includes an amino acid sequence including a region having a locally large hydrophobicity index, which corresponds to substitution of one or more amino acid residues in REP with an amino acid residue having a large hydrophobicity index and/or insertion of one or more amino acid residues having a large hydrophobicity index into REP, as compared with a naturally derived fibroin:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
[25] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [24],
wherein the region having a locally large hydrophobicity index is composed of 2 to 4 consecutive amino acid residues.
[26] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [24] or [25],
wherein the amino acid residue having a large hydrophobicity index is selected from among isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A).
[27] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein, when the total number of amino acid residues included in a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as p, and the total number of amino acid residues included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as q, p/q is 6.2% or more:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
[28] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [24] to [27],
wherein the modified fibroin includes an amino acid sequence corresponding to a case in which one or more amino acid residues in REP are substituted with an amino acid residue having a large hydrophobicity index and/or one or more amino acid residues having a large hydrophobicity index are inserted into REP, and additionally, one or more amino acid residues are substituted, deleted, inserted and/or added, as compared with a naturally derived fibroin.
[29] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif, and
wherein the domain sequence includes an amino acid sequence with a reduced content of glutamine residues, which corresponds to deletion of one or more glutamine residues in REP or substitution with other amino acid residues, as compared with a naturally derived fibroin:
[in Formula 1 and in Formula 2, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 80% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
[30] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [29],
wherein the modified fibroin includes a GPGXX (where, X represents an amino acid residue other than a glycine residue) motif in REP, and the content of the GPGXX motif is 10% or more.
[31] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to [29] or [30],
wherein the modified fibroin has a glutamine residue content of 9% or less.
[32] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [29] to [31],
wherein the other amino acid residue is an amino acid residue selected from the group consisting of isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H).
[33] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [29] to [32],
wherein, in the modified fibroin, the hydrophobicity of REP is -0.8 or more.
[34] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [29] to [33],
wherein the modified fibroin includes an amino acid sequence corresponding to a case in which one or more glutamine residues in REP are deleted or substituted with other amino acid residues and additionally one or more amino acid residues are substituted, deleted, inserted and/or added, as compared with a naturally derived fibroin.
[35] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin has a limit oxygen index (LOI) value of 26.0 or more.
[36] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14],
wherein the modified fibroin has a maximum hygroscopic heat generation of greater than 0.025°C/g determined according to the following Formula A: $Maximum hygroscopic heat generation = \{(maximum value of sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment) - (sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment)\} (° C) / sample weight (g)$
[in Formula A, a low humidity environment means an environment of a temperature of 20°C and a relative humidity of 40%, and a high humidity environment means an environment of a temperature of 20°C and a relative humidity of 90%].

Effects of Invention

According to the present invention, it is possible to provide a high-shrinkage artificial fibroin spun yarn which has a sufficiently high shrinkage rate and an excellent tactile feel and flexibility, and also can be manufactured safely and a method for manufacturing the same. According to the present invention, it is also possible to provide an artificial fibroin spun yarn which allows a high-shrinkage artificial fibroin spun yarn to be manufactured at a sufficiently high shrinkage rate and safely, and a method for shrinking the same.

Brief Description of Drawings

FIG. 1 is a schematic view showing a domain sequence of a modified fibroin according to one embodiment.
FIG. 2 is a diagram showing a distribution of values of z/w (%) of naturally derived fibroin.
FIG. 3 is a diagram showing a distribution of values of x/y (%) of naturally derived fibroin.
FIG. 4 is a schematic view showing a domain sequence of a modified fibroin according to one embodiment.
FIG. 5 is a schematic view showing a domain sequence of a modified fibroin according to one embodiment.
FIG. 6 is an explanatory diagram schematically showing an example of a spinning device for manufacturing modified fibroin fibers (filaments).
FIG. 7 is a graph showing an example of results of a hygroscopic heat generation test.

Description of Embodiments

Forms for implementing the present invention will be described below in detail. However, the present invention is not limited to the following embodiments.
[High-shrinkage artificial fibroin spun yarn] A high-shrinkage artificial fibroin spun yarn according to the present embodiment is a shrunk artificial fibroin fiber containing a modified fibroin. The high-shrinkage artificial fibroin spun yarn according to the present embodiment has a shrinkage rate of greater than 1% defined in the following Formula I. $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$

The modified fibroin according to the present embodiment is a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif. In the modified fibroin, an amino acid sequence (N-terminal sequence and C-terminal sequence) may be additionally added to one or both of the N-terminal side and the C-terminal side of the domain sequence. Although not limited to this, the N-terminal sequence and the C-terminal sequence are typically regions having no repeat of an amino acid motif characteristic of fibroin and are composed of amino acids of about 100 residues.
"Modified fibroin" in this specification means an artificially manufactured fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from an amino acid sequence of a naturally derived fibroin, or may a fibroin having an amino acid sequence the same as that of a naturally derived fibroin. "Naturally derived fibroin" in this specification is also a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, or Formula 2: [(A)_n motif-REP]_m-(A)_n motif.
"Modified fibroin" may be a fibroin in which an amino acid sequence of a naturally derived fibroin is used without change, a fibroin whose amino acid sequence is modified from the amino acid sequence of a naturally derived fibroin (for example, a fibroin whose amino acid sequence is modified by modifying a cloned gene sequence of a naturally derived fibroin), or a fibroin that is artificially designed and synthesized regardless of a naturally derived fibroin (for example, a fibroin having a desired amino acid sequence obtained by chemically synthesizing nucleic acids that encode a designed amino acid sequence). Here, regarding the modified fibroin according to the present embodiment, a modified spider silk fibroin is preferably used because it has excellent heat retention, hygroscopic heat generation characteristics and/or flame retardancy.
"Domain sequence" in this specification refers to an amino acid sequence that generates a crystalline region specific to a fibroin (typically, a region corresponding to the (A)_n motif of an amino acid sequence) and a non-crystalline region (typically, a region corresponding to an REP of an amino acid sequence) and an amino acid sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif. Here, the (A)_n motif represents an amino acid sequence mainly containing alanine residues, and the number of amino acid residues is 2 to 27. The number of amino acid residues in the (A)_n motif is an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. In addition, a ratio of the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). A plurality of (A)_n motifs present in the domain sequence may be composed of only at least 7 alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200, 10 to 180, 10 to 160, 10 to 140, 10 to 120, 10 to 100, 10 to 80, 10 to 60, or 10 to 40 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 8 to 300, 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, 20 to 140, or 20 to 120. A plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.
For example, the modified fibroin according to the present embodiment can be obtained by performing an amino acid sequence modification on a cloned gene sequence of a naturally derived fibroin, for example, corresponding to substitution, deletion, insertion and/or addition of one or more amino acid residues. Amino acid residues can be substituted, deleted, inserted and/or added using methods well known to those skilled in the art such as partial mutagenesis. Specifically, a method described in the document Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983) can be used.
A naturally derived fibroin is a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, or Formula 2: [(A)_n motif-REP]_m-(A)_n motif, and specific examples thereof include fibroins produced by insects or spiders.
Examples of fibroins produced by insects include silk proteins produces by silkworms such as Bombyx mori, Kuwako (Bombyx mandarina), Japanese silk moth (Antheraea yamamai), tussar silkmoth (Anteraea pernyi), maple silkworm (Eriogyna pyretorum), cynthia silkworm (Pilosamia Cynthia ricini), ailanthus silkmoth (Samia cynthia), apanese giant silkworm (Caligura japonica), tasar silkworm (Antheraea mylitta), and muga silkworm (Antheraea assama), and hornet silk proteins that are discharged by larvae such as those of the hornet (Vespa simillima xanthoptera).
More specific examples of fibroins produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).
Examples of fibroins produced by spiders include spider silk proteins produced by spiders belonging to the genus Araneus such as Araneus ventricosus, Araneus diadematus, Araneus pentagrammicus, Araneus pentagrammicus and Araneus nojimai, spiders belonging to the genus Neoscona such as Neoscona scylla, Neoscona nautica, Neoscona adianta and Neoscona scylloides, spiders belonging to the genus Pronus such as Pronoides brunneus, spiders belonging to the genus Cyrtarachne such as Cyrtarachne bufo and Cyrtarachne inaequalis, spiders belonging to the genus Gasteracantha such as Gasteracantha kuhli and Thelacantha brevipina, spiders belonging to the genus Ordgarius such as Ordgarius hobsoni and Ordgarius sexspinosus, spiders belonging to the genus Argiope such as Argiope amoena, Argiope minuta and Argiope bruennichi, spiders belonging to the genus Arachnura such as Arachnura logio, spiders belonging to the genus Acusilas such as Acusilas coccineus, spiders belonging to the genus Cytophora such as Cyrtophora ikomosanensis, Cyrtophora exanthematica and Cyrtophora unicolor, spiders belonging to the genus Poltys such as Poltys illepidus, spiders belonging to the genus Cyclosa such as Cyclosa octotuberculata, Cyclosa sedeculata, Cyclosa vallata and Cyclosa atrata, and spiders belonging to the genus Chorizopes such as Chorizopes nipponicus, and spider silk proteins produced by spiders belonging to the genus Tetragnatha such as Tetragnatha praedonia, Tetragnatha maxillosa, Tetragnatha extensa and Tetragnatha squamata, spiders belonging to the genus Leucauge such as Leucauge celebesiana, Leucauge blanda and Leucauge subblanda, spiders belonging to the genus Nephila such as Nephila clavata and Nephila pilipes, spiders belonging to the genus Menosira such as Menosira ornata, spiders belonging to the genus Dyschiriognatha such as Tetragnathidae, spiders belonging to the genus Latrodectus such as Latrodectus mactans, Latrodectus hasseltii, Latrodectus geometricus and Latrodectus tredecimguttat, and spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline silk proteins such as MaSp (MaSpl and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSpl and MiSp2).
More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3(adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4(adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession number AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession number AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [derived from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), and the like.
More specific examples of naturally derived fibroins may further include fibroins whose sequence information is registered in the NCBI GenBank. For example, within sequence information registered in the NCBI GenBank, from sequences containing INV as DIVISION, sequences in which spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" are used as keywords in DEFINITION, it can be confirmed that a character string of a specific product from CDS, and sequences in which a specific character string is used in TISSUE TYPE from SOURCE are extracted.
The modified fibroin according to the present embodiment may be a modified silk fibroin (in which an amino acid sequence of a silk protein produced by silkworms is modified) or a modified spider silk fibroin (in which an amino acid sequence of a spider silk protein produced by spiders is modified). Regarding the modified fibroin, a modified spider silk fibroin is preferable because it has excellent heat retention, hygroscopic heat generation characteristics and/or flame retardancy.
Specific examples of modified fibroins include a modified fibroin derived from a large vesicular transport silk protein produced in the large ampullar gland of a spider (first modified fibroin), a modified fibroin having a domain sequence with a reduced content of glycine residues (second modified fibroin), a modified fibroin having a domain sequence with a reduced content of the (A)_n motif (third modified fibroin), a modified fibroin with a reduced content of glycine residues and a reduced content of the (A)_n motif (fourth modified fibroin), a modified fibroin having a domain sequence containing a region having a locally high hydrophobicity index (fifth modified fibroin), and a modified fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified fibroin).
Examples of first modified fibroins include a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m. In the first modified fibroin, the number of amino acid residues of the (A)_n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, still more preferably an integer of 10 to 20, still more preferably an integer of 4 to 16, particularly preferably an integer of 8 to 16, and most preferably an integer of 10 to 16. In the first modified fibroin, in Formula 1, the number of amino acid residues constituting REP is preferably 10 to 200, more preferably 10 to 150, still more preferably 20 to 100, and still more preferably 20 to 75. In the first modified fibroin, a total number of glycine residues, serine residues and alanine residues included in the amino acid sequence represented by Formula 1: [(A)_n motif-REP]_m is preferably 40% or more, more preferably 60% or more, and still more preferably 70% or more with respect to the total number of amino acid residues.
The first modified fibroin includes a unit of the amino acid sequence represented by Formula 1: [(A)_n motif-REP]_m and may be a polypeptide in which the C-terminal sequence is an amino acid sequence shown in any of SEQ ID NOs 1 to 3 or an amino acid sequence having a homology of 90% or more with an amino acid sequence shown in any of SEQ ID NOs 1 to 3.
The amino acid sequence shown in SEQ ID NO 1 is the same as the amino acid sequence composed of amino acids of 50 residues of the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), the amino acid sequence shown in SEQ ID NO 2 is the same as the amino acid sequence obtained by removing 20 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO 1, and the amino acid sequence shown in SEQ ID NO 3 is the same as the amino acid sequence obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO 1.
More specific examples of first modified fibroins include a modified fibroin including (1-i) an amino acid sequence shown in SEQ ID NO 4 (recombinant spider silk protein ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO 4. The sequence identity is preferably 95% or more.
The amino acid sequence shown in SEQ ID NO 4 is obtained by performing mutation on the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO 5) composed of the start codon, His10 tag and HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal such that the number of 1st to 13th repeat regions is approximately doubled, and the translation is terminated at the 1,154th amino acid residue. The amino acid sequence of the C-terminal of the amino acid sequence shown in SEQ ID NO 4 is the same as the amino acid sequence shown in SEQ ID NO 3.
The modified fibroin (1-i) may be composed of the amino acid sequence shown in SEQ ID NO 4.
As compared with a naturally derived fibroin, in the second modified fibroin, the domain sequence includes an amino acid sequence with a reduced content of glycine residues. It can be said that the second modified fibroin has an amino acid sequence corresponding to a case in which at least one or more glycine residues in REP are substituted with other amino acid residues, as compared with a naturally derived fibroin.
As compared with a naturally derived fibroin, in the second modified fibroin, the domain sequence includes an amino acid sequence corresponding to a case in which, in at least one motif sequence selected from among GGX and GPGXX (where, G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP, one glycine residue in at least one or more motif sequences is substituted with another amino acid residue.
In the second modified fibroin, a proportion of the motif sequences in which the above glycine residue is substituted with another amino acid residue with respect to all motif sequences may be 10% or more.
The second modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and may include an amino acid sequence in which, when the total number of amino acid residues of amino acid sequences composed of XGX (where, X represents an amino acid residue other than glycine) contained in all REPs in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as z, and the total number of amino acid residues in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as w, z/w is 30% or more, 40% or more, 50% or more or 50.9% or more. The number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and still more preferably 100% (meaning that it is composed of only alanine residues).
The second modified fibroin is preferably a modified fibroin in which the proportional content of amino acid sequences composed of XGX is increased by substituting one glycine residue of a GGX motif with another amino acid residue. In the second modified fibroin, the proportional content of amino acid sequences composed of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, still more preferably 10% or less, still more preferably 6% or less, still more preferably 4% or less, and particularly preferably 2% or less. The proportional content of amino acid sequences composed of GGX in the domain sequence can be calculated by the same method as the following method for calculating the proportional content (z/w) of the amino acid sequences composed of XGX.
A method for calculating z/w will be described in more detail. First, in a fibroin (modified fibroin or naturally derived fibroin) including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, an amino acid sequence composed of XGX is extracted from all REPs included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences composed of XGX are extracted (no overlap), z is 50x3=150. In addition, for example, when there is X contained in two XGXs (X at the center), as in the case of the amino acid sequence composed of XGXGX, calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues). w is the total number of amino acid residues contained in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4+50+4+100+4+10+4+20+4+30=230 (the (A)_n motif positioned closest to the C-terminal side is excluded). Next, z/w (%) can be calculated by dividing z by w.
Here, z/w in the naturally derived fibroin will be described. First, as described above, according to confirmation using the exemplified method in which a fibroin whose amino acid sequence information is registered in the NCBI GenBank is extracted, 663 types of fibroins (of which 415 types are fibroins produced by spiders) are extracted. Among all of the extracted fibroins, z/w is calculated from the amino acid sequence of the naturally derived fibroin including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m and in which the proportional content of amino acid sequence composed of GGX in the fibroin is 6% or less by the above calculation method. The results are shown in FIG. 2. In FIG. 2, the horizontal axis represents z/w (%), and the vertical axis represents frequency. As can be clearly understood from FIG. 2, z/w in the naturally derived fibroin is always less than 50.9% (the highest is 50.86%).
In the second modified fibroin, z/w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, still more preferably 70% or more, and still more preferably 80% or more. The upper limit of z/w is not particularly limited, and may be, for example, 95% or less.
For example, the second modified fibroin can be obtained by performing a modification on a cloned gene sequence of the naturally derived fibroin such that at least a part of the nucleotide sequence that encodes a glycine residue is substituted and encodes another amino acid residue. In this case, regarding the glycine residue to be modified, one glycine residue in a GGX motif and a GPGXX motif may be selected or substitution may be performed so that z/w is 50.9% or more. In addition, for example, the second modified fibroin can be obtained by designing an amino acid sequence that meets the requirements for the above aspect using the amino acid sequence of a naturally derived fibroin and chemically synthesizing nucleic acids that encode the designed amino acid sequence. In any case, in addition to a modification in which a glycine residue in REP is substituted with another amino acid residue from the amino acid sequence of a naturally derived fibroin, the amino acid sequence may be additionally modified such as by substitution, deletion, insertion and/or addition of one or more amino acid residues.
The above other amino acid residues are not particularly limited as long as they are amino acid residues other than glycine residues, and hydrophobic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, methionine (M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W) residues, and hydrophilic amino acid residue such as glutamine (Q) residues, asparagine (N) residues, serine (S) residues, lysine (K) residues and glutamic acid (E) residues are preferable, valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine (F) residues and glutamine (Q) residues are more preferable, and glutamine (Q) residues are still more preferable.
More specific examples of second modified fibroins include a modified fibroin including (2-i) an amino acid sequence shown in SEQ ID NO 6 (Met-PRT380), SEQ ID NO 7 (Met-PRT410), SEQ ID NO 8 (Met-PRT525) or SEQ ID NO 9 (Met-PRT799) or (2-ii) an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
The modified fibroin (2-i) will be described. In the amino acid sequence shown in SEQ ID NO 6, all GGXs in REP of the amino acid sequence shown in SEQ ID NO 10 (Met-PRT313) corresponding to the naturally derived fibroin are substituted with GQX. In the amino acid sequence shown in SEQ ID NO 7, every two (A)_n motifs from the N-terminal side to the C-terminal side are deleted from the amino acid sequence shown in SEQ ID NO 6, and additionally, one [(A)_n motif-REP] is inserted before the C-terminal sequence. In the amino acid sequence shown in SEQ ID NO 8, two alanine residues are inserted into the C-terminal side of each (A)_n motif of the amino acid sequence shown in SEQ ID NO 7, and additionally, some glutamine (Q) residues are substituted with serine (S) residues, and some amino acids on the C-terminal side are deleted so that the molecular weight is almost the same as that of SEQ ID NO 7. In the amino acid sequence shown in SEQ ID NO 9, a predetermined hinge sequence and His tag sequence are added to the C-terminal of a sequence obtained by repeating a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO 7 (where, several amino acid residues on the C-terminal side of the region are substituted) four times.
The value of z/w in the amino acid sequence shown in SEQ ID NO 10 (corresponding to the naturally derived fibroin) is 46.8%. The values of z/w in the amino acid sequence shown in SEQ ID NO 6, the amino acid sequence shown in SEQ ID NO 7, the amino acid sequence shown in SEQ ID NO 8, and the amino acid sequence shown in SEQ ID NO 9 are 58.7%, 70.1%, 66.1% and 70.0%, respectively. In addition, the values of x/y at the Giza ratio (to be described below) of 1:1.8 to 11.3 in the amino acid sequences shown in SEQ ID NO 10, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9 are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
The modified fibroin (2-i) may be composed of an amino acid sequence shown in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
The modified fibroin (2-ii) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9. The modified fibroin (2-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin (2-ii) has a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, and when the total number of amino acid residues of the amino acid sequence composed of XGX (where, X represents an amino acid residue other than glycine) contained in REP is set as z, and the total number of amino acid residues of REP in the above domain sequence is set as w, z/w is preferably 50.9% or more.
The second modified fibroin may include a tag sequence at either or both of the N-terminal and the C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
Regarding the tag sequence, for example, an affinity tag using specific affinity (binding property, affinity) with other molecules may be exemplified. Specific examples of affinity tags include a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are present and has a property of specifically binding to metal ions such as nickel, and thus can be used for isolating a modified fibroin through chelating metal chromatography. Specific examples of tag sequences include, for example, the amino acid sequence shown in SEQ ID NO 11 (an amino acid sequence including a His tag sequence and a hinge sequence).
In addition, a tag sequence such as glutathione-S-transferase (GST) that specifically binds to glutathione and a maltose binding protein (MBP) that specifically binds to maltose can be used.
In addition, an "epitope tag" using an antigen-antibody reaction can be used. When a peptide (epitope) exhibiting antigenicity is added as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include an HA (a peptide sequence of hemagglutinin of influenza virus) tag, a myc tag, and a FLAG tag. When an epitope tag is used, the modified fibroin can be easily purified with high specificity.
In addition, a tag sequence that can be disconnected with a specific protease can be used. When a protein adsorbed via the tag sequence is treated with a protease, it is possible to collect the modified fibroin from which the tag sequence is disconnected.
More specific examples of modified fibroins containing a tag sequence include a modified fibroin including (2-iii) an amino acid sequence shown in SEQ ID NO 12 (PRT380), SEQ ID NO 13 (PRT410), SEQ ID NO 14 (PRT525) or SEQ ID NO 15 (PRT799), or (2-iv) an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
The amino acid sequences shown in SEQ ID NO 16 (PRT313), SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15 are obtained by adding the amino acid sequence shown in SEQ ID NO 11 (including a His tag sequence and a hinge sequence) to the N-terminal of the amino acid sequences shown in SEQ ID NO 10, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, respectively.
The modified fibroin (2-iii) may be composed of an amino acid sequence shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
The modified fibroin (2-iv) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15. The modified fibroin (2-iv) is also a protein including 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 a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15, and when the total number of amino acid residues of the amino acid sequence composed of XGX (where, X represents an amino acid residue other than glycine) contained in REP is set as z, and the total number of amino acid residues of REP in the above domain sequence is set as w, z/w is preferably 50.9% or more.
The second modified fibroin may include a secretory signal for releasing the protein produced in a recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
As compared with a naturally derived fibroin, in the third modified fibroin, the domain sequence includes an amino acid sequence with a reduced content of the (A)_n motif. It can be said that the domain sequence of the third modified fibroin includes an amino acid sequence corresponding to a case in which at least one or more (A)_n motifs are deleted, as compared with a naturally derived fibroin.
The third modified fibroin may include an amino acid sequence corresponding to a case in which 10 to 40% of the (A)_n motif is deleted from the naturally derived fibroin.
As compared with a naturally derived fibroin, in the third modified fibroin, the domain sequence may include an amino acid sequence corresponding to a case in which at least one (A)_n motif for every one to three (A)_n motifs from the N-terminal side to the C-terminal side is deleted.
As compared with a naturally derived fibroin, in the third modified fibroin, the domain sequence may include an amino acid sequence corresponding to a case in which, from the N-terminal side to the C-terminal side, at least deletion of two consecutive (A)_n motifs and deletion of one (A)_n motif are repeated in that order.
In the third modified fibroin, the domain sequence may include an amino acid sequence corresponding to a case in which at least every two (A)_n motifs are deleted from the N-terminal side to the C-terminal side.
The third modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and may include an amino acid sequence in which, when the numbers of amino acid residues of REP of adjacent two [(A)_n motif-REP] units are sequentially compared from the N-terminal side to the C-terminal side, the maximum value of the total value obtained by adding the number of amino acid residues of the adjacent two [(A)_n motif-REP] units in which a ratio of the number of amino acid residues of the other REP with respect to 1 set for the number of amino acid residues of one REP with a smaller number of amino acid residues is 1.8 to 11.3 is set as x, and the total number of amino acid residues of the domain sequence is set as y, x/y is 20% or more, 30% or more, 40% or more or 50% or more. The number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif may be 83% or more, and is preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and still more preferably 100% (meaning it is composed of only alanine residues).
A method for calculating x/y will be described in more detail with reference to FIG. 1. FIG. 1 shows a domain sequence in which the N-terminal sequence and the C-terminal sequence are removed from the modified fibroin. The domain sequence includes a sequence of (A)_n motif-first REP (50 amino acid residues)-(A)_n motif-second REP (100 amino acid residues)-(A)_n motif-third REP (10 amino acid residues)-(A)_n motif-fourth REP (20 amino acid residues)-(A)_n motif-fifth REP (30 amino acid residues)-(A)_n motif from the N-terminal side (left side).
Adjacent two [(A)_n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so that there is no overlap. In this case, an unselected [(A)_n motif-REP] unit may be present. FIG. 1 shows Pattern 1 (comparison of a first REP and a second REP, and comparison of a third REP and a fourth REP), Pattern 2 (comparison of a first REP and a second REP, and comparison of a fourth REP and a fifth REP), Pattern 3 (comparison of a second REP and a third REP and comparison of a fourth REP and a fifth REP), and Pattern 4 (comparison of a first REP and a second REP). Here, there are other selection methods.
Next, in the patterns, the numbers of amino acid residues of each REP in the selected adjacent two [(A)_n motif-REP] units are compared. The comparison is performed by calculating a ratio of the number of amino acid residues of the other REP with respect to 1 set for one REP with a smaller number of amino acid residues. For example, in the case of comparison of the first REP (50 amino acid residues) and the second REP (100 amino acid residues), a ratio of the number of amino acid residues of the second REP with respect to 1 set for the first REP having a smaller number of amino acid residues is 100/50=2. Similarly, in the case of comparison of the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), a ratio of the number of amino acid residues of the fifth REP with respect to 1 set for the fourth REP with a smaller number of amino acid residues is 30/20= 1.5.
In FIG. 1, a set of [(A)_n motif-REP] units in which a ratio of the number of amino acid residues of the other REP with respect to 1 set for one REP with a smaller number of amino acid residues is 1.8 to 11.3 is indicated by a solid line. In this specification, this ratio is called a Giza ratio. A set of [(A)_n motif-REP] units in which a ratio of the number of amino acid residues of the other REP with respect to 1 set for one REP with a smaller number of amino acid residues is less than 1.8 or more than 11.3 is indicated by a dotted line.
In the patterns, the total number of amino acid residues of adjacent two [(A)_n motif-REP] units indicated by a solid line is summed (not only REP but also the number of amino acid residues in the (A)_n motif). Then, the sum total values are compared, and a total value (a maximum value of the total value) of the pattern in which the total value is a maximum is set as x. In the example shown in FIG. 1, the total value of Pattern 1 is a maximum.
Next, x/y (%) can be calculated by dividing x by the total number y of amino acid residues of the domain sequence.
In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, still more preferably 70% or more, still more preferably 75% or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the Giza ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, when the Giza ratio is 1:1.8 to 3.4, x/y is preferably 77.1% or more, when the Giza ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and when the Giza ratio is 1:1.9 to 4.1, x/y is preferably 64.2% or more.
When the third modified fibroin is a modified fibroin in which at least seven of the plurality of (A)_n motifs present in the domain sequence are composed of only alanine residues, x/y is preferably 46.4% or more, more preferably 50% or more, still more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly limited, and may be 100% or less.
Here, x/y in the naturally derived fibroin will be described. First, as described above, according to confirmation using the exemplified method in which a fibroin whose amino acid sequence information is registered in the NCBI GenBank is extracted, 663 types of fibroins (of which 415 types are fibroins produced by spiders) are extracted. Among all of the extracted fibroins, x/y is calculated from the amino acid sequence of the naturally derived fibroin composed of a domain sequence represented by Formula 1: [(A)_n motif-REP]_m according to the above calculation method. The results when the Giza ratio is 1:1.9 to 4.1 are shown in FIG. 3.
In FIG. 3, the horizontal axis represents x/y (%), and the vertical axis represents frequency. As can be clearly understood from FIG. 3, x/y in the naturally derived fibroin is always less than 64.2% (the highest is 64.14%).
For example, the third modified fibroin can be obtained by deleting one or more sequences that encode the (A)_n motif from the cloned gene sequence of the naturally derived fibroin so that x/y is 64.2% or more. In addition, for example, an amino acid sequence corresponding to a case in which one or more (A)_n motifs are deleted from the amino acid sequence of the naturally derived fibroin so that x/y is 64.2% or more may be designed, and nucleic acids that encode the designed amino acid sequence may be chemically synthesized to obtain the third modified fibroin. In any case, in addition to a modification in which the (A)_n motif is deleted from the amino acid sequence of the naturally derived fibroin, the amino acid sequence may be additionally modified such as by substitution, deletion, insertion and/or addition of one or more amino acid residues.
More specific examples of third modified fibroins include a modified fibroin including (3-i) an amino acid sequence shown in SEQ ID NO 17 (Met-PRT399), SEQ ID NO 7 (Met-PRT410), SEQ ID NO 8 (Met-PRT525) or SEQ ID NO 9 (Met-PRT799) or (3-ii) an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
The modified fibroin (3-i) will be described. In the amino acid sequence shown in SEQ ID NO 17, every two (A)_n motifs from the N-terminal side to the C-terminal side are deleted from the amino acid sequence shown in SEQ ID NO 10 (Met-PRT313) corresponding to the naturally derived fibroin, and additionally, one [(A)_n motif-REP] is inserted before the C-terminal sequence. The amino acid sequences shown in SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9 are the same as those described in the second modified fibroin.
The value of x/y at the Giza ratio of 1:1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO 10 (corresponding to the naturally derived fibroin) is 15.0%. The values of x/y in the amino acid sequence shown in SEQ ID NO 17 and the amino acid sequence shown in SEQ ID NO 7 are always 93.4%. The value of x/y in the amino acid sequence shown in SEQ ID NO 8 is 92.7%. The value of x/y in the amino acid sequence shown in SEQ ID NO 9 is 89.8%. The values of z/w in the amino acid sequences shown in SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9 are 46.8%, 56.2%, 70.1%, 66.1% and 70.0%, respectively.
The modified fibroin (3-i) may be composed of an amino acid sequence shown in SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
The modified fibroin (3-ii) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9. The modified fibroin (3-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin (3-ii) has a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, and when the numbers of amino acid residues of REP of adjacent two [(A)_n motif-REP] units from the N-terminal side to the C-terminal side are sequentially compared, the maximum value of the total value obtained by adding the number of amino acid residues of the adjacent two [(A)_n motif-REP] units in which a ratio of the number of amino acid residues of the other REP with respect to 1 set for the number of amino acid residues of one REP with a smaller number of amino acid residues is 1.8 to 11.3 (the Giza ratio is 1:1.8 to 11.3) is set as x, and the total number of amino acid residues of the domain sequence is set as y, x/y is preferably 64.2% or more.
The third modified fibroin may include the above tag sequence at either or both of the N-terminal and the C-terminal.
More specific examples of modified fibroins including a tag sequence include a modified fibroin including (3-iii) an amino acid sequence shown in SEQ ID NO 18 (PRT399), SEQ ID NO 13 (PRT410), SEQ ID NO 14 (PRT525) or SEQ ID NO 15 (PRT799) or (3-iv) an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
The amino acid sequences shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15 are obtained by adding the amino acid sequence shown in SEQ ID NO 11 (including a His tag sequence and a hinge sequence) of the N-terminal of the amino acid sequences shown in SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, respectively.
The modified fibroin (3-iii) may be composed of an amino acid sequence shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
The modified fibroin (3-iv) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15. The modified fibroin (3-iv) is also a protein including 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 a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15, and when the numbers of amino acid residues of REP of adjacent two [(A)_n motif-REP] units from the N-terminal side to the C-terminal side are sequentially compared, the maximum value of the total value obtained by adding the number of amino acid residues of the adjacent two [(A)_n motif-REP] units in which a ratio of the number of amino acid residues of the other REP with respect to 1 set for the number of amino acid residues of one REP with a smaller number of amino acid residues is 1.8 to 11.3 is set as x, and the total number of amino acid residues of the domain sequence is set as y, x/y is preferably 64.2% or more.
The third modified fibroin may include a secretory signal for releasing the protein produced in a recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
As compared with a naturally derived fibroin, in the fourth modified fibroin, the domain sequence includes an amino acid sequence in which the content of the (A)_n motif is reduced and also the content of glycine residues is reduced. As compared with a naturally derived fibroin, it can be said that the domain sequence of the fourth modified fibroin includes an amino acid sequence corresponding to a case in which at least one or more (A)_n motifs are deleted, and additionally, at least one or more glycine residues in REP are substituted with other amino acid residues. That is, the fourth modified fibroin is a modified fibroin having characteristics of the above second modified fibroin and the third modified fibroin in combination. Specific forms and the like are the same as those described in the second modified fibroin and the third modified fibroin.
More specific examples of fourth modified fibroins include a modified fibroin including (4-i) an amino acid sequence shown in SEQ ID NO 7 (Met-PRT410), SEQ ID NO 8 (Met-PRT525), SEQ ID NO 9 (Met-PRT799), SEQ ID NO 13 (PRT410), SEQ ID NO 14 (PRT525) or SEQ ID NO 15 (PRT799) or (4-ii) an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15. Specific forms of modified fibroins including an amino acid sequence shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15 are as described above.
As compared with a naturally derived fibroin, in the fifth modified fibroin, the domain sequence may include an amino acid sequence corresponding to a case in which one or more amino acid residues in REP are substituted with an amino acid residue having a large hydrophobicity index and/or one or more amino acid residues having a large hydrophobicity index are inserted into REP and containing a region having a locally large hydrophobicity index.
The region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
The above amino acid residue having a large hydrophobicity index is more preferably an amino acid residue selected from among isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A).
As compared with a naturally derived fibroin, in the fifth modified fibroin, in addition to a modification in which one or more amino acid residues in REP are substituted with an amino acid residue having a large hydrophobicity index and/or one or more amino acid residues having a large hydrophobicity index are inserted into REP, as compared with a naturally derived fibroin, the amino acid sequence may be additionally modified such as by substitution, deletion, insertion and/or addition of one or more amino acid residues.
For example, the fifth modified fibroin can be obtained by substituting one or more hydrophilic amino acid residues (for example, amino acid residues with a negative hydrophobicity index) in REP from the cloned gene sequence of the naturally derived fibroin with hydrophobic amino acid residues (for example, amino acid residues with a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid residues into REP. In addition, for example, the fifth modified fibroin can be obtained by designing an amino acid sequence in which one or more hydrophilic amino acid residues in REP from the amino acid sequence of the naturally derived fibroin are substituted with hydrophobic amino acid residues and/or one or more hydrophobic amino acid residues are inserted into REP, and chemically synthesizing nucleic acids that encode the designed amino acid sequence. In any case, in addition to a modification in which one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of the naturally derived fibroin and/or one or more hydrophobic amino acid residues are inserted into REP, the amino acid sequence may be additionally modified such as by substitution, deletion, insertion and/or addition of one or more amino acid residues.
The fifth modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and may include an amino acid sequence in which, when the total number of amino acid residues included in a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as p, and the total number of amino acid residues included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as q, p/q is 6.2% or more.

Regarding the hydrophobicity index of the amino acid residue, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein," J. Mol. Biol., 157, pp. 105-132) may be used. Specifically, the hydrophobicity index (hydropathy index, hereinafter referred to as "HI") of each amino acid is as shown in the following Table 1.

[Table 1]

Amino acid	HI	Amino acid	HI
Isoleucine (Ile)	4.5	Tryptophan (Trp)	-0.9
Valine (Val)	4.2	Tyrosine (Tyr)	-1.3
Leucine (Leu)	3.8	Proline (Pro)	-1.6
Phenylalanine (Phe)	2.8	Histidine (His)	-3.2
Cysteine (Cys)	2.5	Asparagine (Asn)	-3.5
Methionine (Met)	1.9	Aspartic acid (Asp)	-3.5
Alanine (Ala)	1.8	Glutamine (Gln)	-3.5
Glycine (Gly)	-0.4	Glutamic acid (Glu)	-3.5
Threonine (Thr)	-0.7	Lysine (Lys)	-3.9
Serine (Ser)	-0.8	Arginine (Arg)	-4.5

A method for calculating p/q will be described in more detail. For calculation, the sequence (hereinafter referred to as a "sequence A") obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by Formula 1: [(A)_n motif-REP]_m is used. First, in all REPs included in the sequence A, the average value of the hydrophobicity index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of HI of amino acid residues included in 4 consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all 4 consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if some amino acid residues correspond to a plurality of "4 consecutive amino acid residues in which the average value of the hydrophobicity index is 2.6 or more," they are included in the region as one amino acid residue. Thus, the total number of amino acid residues included in the region is p. In addition, the total number of amino acid residues included in the sequence A is q.
For example, when 20 "4 consecutive amino acid residues in which the average value of the hydrophobicity index is 2.6 or more" are extracted (no overlap), 20 of 4 consecutive amino acid residues (no overlap) are included in the region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more, and p is 20x4=80. In addition, for example, when two "4 consecutive amino acid residues in which the average value of the hydrophobicity index is 2.6 or more" overlap by one amino acid residue, 7 amino acid residues are included in the region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more (p=2×4-1=7, "-1" is deduction for overlap). For example, in the case of the domain sequence shown in FIG. 4, since there are seven "4 consecutive amino acid residues in which the average value of the hydrophobicity index is 2.6 or more" without overlap, p is 7x4=28. In addition, for example, in the case of the domain sequence shown in FIG. 4, q is 4+50+4+40+4+10+4+20+4+30=170 ((A)_n motif present at the end of the C-terminal side is not included). Next, p/q (%) can be calculated by dividing p by q. In FIG. 4, 28/170=16.47%.
In the fifth modified fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, still more preferably 20% or more, and still more preferably 30% or more. The upper limit of p/q is not particularly limited, and may be, for example, 45% or less.
For example, the fifth modified fibroin can be obtained by performing a modification on a cloned amino acid sequence of the naturally derived fibroin such that one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in REP are substituted with hydrophobic amino acid residues (for example, amino acid residues having a positive hydrophobicity index) and/or one or more hydrophobic amino acid residues are inserted into REP so that the above p/q condition is satisfied, and thus a modification to an amino acid sequence including a region having a locally large hydrophobicity index is performed. In addition, for example, the fifth modified fibroin can be obtained by designing an amino acid sequence that satisfies the above p/q condition from the amino acid sequence of the naturally derived fibroin, and chemically synthesizing nucleic acids that encode the designed amino acid sequence. In any case, as compared with a naturally derived fibroin, in addition to a modification in which one or more amino acid residues in REP are substituted with an amino acid residue having a large hydrophobicity index and/or one or more amino acid residues having a large hydrophobicity index are inserted into REP, a modification such as substitution, deletion, insertion and/or addition of one or more amino acid residues are may be additionally performed.
The amino acid residue having a large hydrophobicity index is not particularly limited, and is preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) or alanine (A), and more preferably valine (V), leucine (L) or isoleucine (I).
More specific examples of fifth modified fibroins include a modified fibroin including (5-i) an amino acid sequence shown in SEQ ID NO 19 (Met-PRT720), SEQ ID NO 20 (Met-PRT665) or SEQ ID NO 21 (Met-PRT666) or (5-ii) an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 19, SEQ ID NO 20 or SEQ ID NO 21.
The modified fibroin (5-i) will be described. The amino acid sequence shown in SEQ ID NO 19 is obtained by, with respect to the amino acid sequence shown in SEQ ID NO 7 (Met-PRT410), removing a domain sequence at the terminal of the C-terminal side, inserting two amino acid sequences (VLI) each composed of three amino acid residues into every one REP, additionally, substituting some glutamine (Q) residues with serine (S) residues, and deleting a part of the amino acid on the C-terminal side. The amino acid sequence shown in SEQ ID NO 20 is obtained by inserting one amino acid sequence (VLI) composed of three amino acid residues into every one REP of the amino acid sequence shown in SEQ ID NO 8 (Met-PRT525). The amino acid sequence shown in SEQ ID NO 21 is obtained by inserting two amino acid sequences (VLI) each composed of three amino acid residues into every REP of the amino acid sequence shown in SEQ ID NO 8.
The modified fibroin (5-i) may be composed of an amino acid sequence shown in SEQ ID NO 19, SEQ ID NO 20 or SEQ ID NO 21.
The modified fibroin (5-ii) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 19, SEQ ID NO 20 or SEQ ID NO 21. The modified fibroin (5-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin (5-ii) has a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 19, SEQ ID NO 20 or SEQ ID NO 21, and in all REPs included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, when the total number of amino acid residues included in a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is set as p, and the total number of amino acid residues included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as q, p/q is preferably 6.2% or more.
The fifth modified fibroin may include a tag sequence at either or both of the N-terminal and the C-terminal.
More specific examples of modified fibroins including a tag sequence include a modified fibroin including (5-iii) an amino acid sequence shown in SEQ ID NO 22 (PRT720), SEQ ID NO 23 (PRT665) or SEQ ID NO 24 (PRT666) or (5-iv) an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 22, SEQ ID NO 23 or SEQ ID NO 24.
The amino acid sequences shown in SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24 are obtained by adding the amino acid sequence shown in SEQ ID NO 11 (including a His tag sequence and a hinge sequence) of the N-terminal of the amino acid sequences shown in SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21, respectively.
The modified fibroin (5-iii) may be composed of an amino acid sequence shown in SEQ ID NO 22, SEQ ID NO 23 or SEQ ID NO 24.
The modified fibroin (5-iv) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 22, SEQ ID NO 23 or SEQ ID NO 24. The modified fibroin (5-iv) is also a protein including 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 a sequence identity of a 90% or more with an amino acid sequence shown in SEQ ID NO 22, SEQ ID NO 23 or SEQ ID NO 24, and in all REPs included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, when the total number of amino acid residues included in a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is set as p, and the total number of amino acid residues included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as q, p/q is preferably 6.2% or more.
The fifth modified fibroin may include a secretory signal for releasing the protein produced in a recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The sixth modified fibroin includes an amino acid sequence in which the content of glutamine residues is reduced as compared with a naturally derived fibroin.
The sixth modified fibroin preferably includes at least one motif selected from among a GGX motif and a GPGXX motif in the amino acid sequence of REP.
When the sixth modified fibroin includes a GPGXX motif in REP, the content of the GPGXX motif is generally 1% or more, and may be 5% or more and is preferably 10% or more. The upper limit of the content of the GPGXX motif is not particularly limited, and may be 50% or less or 30% or less.
In this specification, the "content of the GPGXX motif" is a value calculated by the following method.
In a fibroin (a modified fibroin or a naturally derived fibroin) including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif, in all REPs included in the sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, when a number obtained by tripling the total number of GPGXX motifs included in the region (that is, corresponding to the total number of G and P in the GPGXX motif) is set as s, and the total number of amino acid residues of all REPs in which the sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and the (A)_n motif is additionally removed is set as t, the content of the GPGXX motif is calculated as s/t.
In calculation of the content of the GPGXX motif, the "sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" is set as a target because a sequence having a low correlation with the characteristic sequence of the fibroin is included in the "sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence" (sequence corresponding to REP), and when m is small (that is, when the domain sequence is short), since this affects the calculation result of the content of the GPGXX motif, the setting is performed for eliminating this effect. Here, when the "GPGXX motif" is positioned at the C-terminal of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA."
FIG. 5 is a schematic view showing a domain sequence of a modified fibroin. A method for calculating a content of the GPGXX motif will be described in detail with reference to FIG. 5. First, in the domain sequence of the modified fibroin shown in FIG. 5 ("[(A)_n motif-REP]_m-(A)_n motif" type), since all REPs are included in the "sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (in FIG. 5, a sequence shown in a "region A"), the number of GPGXX motifs for calculating s is 7, and s is 7×3=21. Similarly, since all REPs are included in the "sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (in FIG. 5, the sequence shown in the "region A"), the total number t of amino acid residues of all REPs in which the (A)_n motif is additionally removed from the sequence is 50+40+10+20+30=150. Then, s/t (%) can be calculated by dividing s by t, and is 21/150=14.0% in the case of the modified fibroin in FIG. 5.
The glutamine residue content of the sixth modified fibroin is preferably 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%.
In this specification, the "content of glutamine residues" is a value that is calculated by the following method.
In a fibroin (a modified fibroin or a naturally derived fibroin) including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif, in all REPs included in the sequence (the sequence corresponding to the "region A" in FIG. 5) obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of glutamine residues included in this region is set as u, and the total number of amino acid residues of all REPs in which the sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and the (A)_n motif is additionally removed is set as t, the content of glutamine residues is calculated as u/t. In calculation of the content of glutamine residues, the reason why the "sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" is set as a target is the same as above.
As compared with a naturally derived fibroin, in the sixth modified fibroin, the domain sequence may include an amino acid sequence corresponding to a case in which one or more glutamine residues in REP are deleted or substituted with other amino acid residues.
The "other amino acid residue" may be any amino acid residue other than the glutamine residues, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of the amino acid residues is as shown in Table 1.
As shown in Table 1, regarding the amino acid residue having a larger hydrophobicity index than the glutamine residue, amino acid residues selected from among isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M)alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H) may be exemplified. Among these, an amino acid residue selected from among isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable, and an amino acid residue selected from among isoleucine (I), valine (V), leucine (L) and phenylalanine (F) is still more preferable.
In the sixth modified fibroin, the hydrophobicity of REP is preferably -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, still more preferably 0.3 or more, and particularly preferably 0.4 or more. The upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less or 0.7 or less.
In this specification, the "hydrophobicity of REP" is a value calculated by the following method.
In a fibroin (a modified fibroin or a naturally derived fibroin) including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif, in all REPs included in the sequence (the sequence corresponding to the "region A" in FIG. 5) obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, when the sum of the hydrophobicity indexes of the amino acid residues in this region is set as v, and the total number of amino acid residues of all REPs in which the sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and the (A)_n motif is additionally removed is set as t, the hydrophobicity of REP is calculated as v/t. In calculation of hydrophobicity of REP, the reason why the "sequence obtained by removing a sequence from the (A)_n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" is set as a target is the same as above.
As compared with a naturally derived fibroin, in the sixth modified fibroin, in the domain sequence, in addition to a modification in which one or more glutamine residues in REP are deleted and/or one or more glutamine residues in REP are substituted with other amino acid residues, the amino acid sequence may be additionally modified such as by substitution, deletion, insertion and/or addition of one or more amino acid residues.
For example, the sixth modified fibroin can be obtained by deleting one or more glutamine residues in REP from the cloned gene sequence of the naturally derived fibroin and/or substituting one or more glutamine residues in REP with other amino acid residues. In addition, for example, the sixth modified fibroin can be obtained by designing an amino acid sequence corresponding to a case in which one or more glutamine residues in REP are deleted from the amino acid sequence of the naturally derived fibroin and/or one or more glutamine residues in REP are substituted with other amino acid residues and chemically synthesizing nucleic acids that encode the designed amino acid sequence.
More specific examples of sixth modified fibroins include (6-i) a modified fibroin including an amino acid sequence shown in SEQ ID NO 25 (Met-PRT888), SEQ ID NO 26 (Met-PRT965), SEQ ID NO 27 (Met-PRT889), SEQ ID NO 28 (Met-PRT916), SEQ ID NO 29 (Met-PRT918), SEQ ID NO 30 (Met-PRT699), SEQ ID NO 31 (Met-PRT698) or SEQ ID NO 32 (Met-PRT966) or (6-ii) a modified fibroin including an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 or SEQ ID NO 32.
The modified fibroin (6-i) will be described. The amino acid sequence shown in SEQ ID NO 25 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO 7 (Met-PRT410) with VL. The amino acid sequence shown in SEQ ID NO 26 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO 7 with TS and substituting the remaining Q with A. The amino acid sequence shown in SEQ ID NO 27 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO 7 with VL and substituting the remaining Q with I. The amino acid sequence shown in SEQ ID NO 28 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO 7 with VI and substituting the remaining Q with L. The amino acid sequence shown in SEQ ID NO 29 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO 7 with VF and substituting the remaining Q with I.
The amino acid sequence shown in SEQ ID NO 30 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO 8 (Met-PRT468) with VL. The amino acid sequence shown in SEQ ID NO 31 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO 8 with VL and substituting the remaining Q with I.
The amino acid sequence shown in SEQ ID NO 32 is obtained by substituting all QQs in a sequence in which a region including 20 domain sequences in the amino acid sequence shown in SEQ ID NO 7 (Met-PRT410) is repeated twice with VF and substituting the remaining Q with I.

All of the amino acid sequences shown in SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32 have a glutamine residue content of 9% or less (Table 2).

[Table 2]

Modified fibroin	Glutamine residue content	GPGXX motif content	Hydrophobicity of REP
Met-PRT410 (SEQ ID NO 7)	17.7%	27.9%	-1.52
Met-PRT888 (SEQ ID NO 25)	6.3%	27.9%	-0.07
Met-PRT965 (SEQ ID NO 26)	0.0%	27.9%	-0.65
Met-PRT889 (SEQ ID NO 27)	0.0%	27.9%	0.35
Met-PRT916 (SEQ ID NO 28)	0.0%	27.9%	0.47
Met-PRT918 (SEQ ID NO 29)	0.0%	27.9%	0.45
Met-PRT699 (SEQ ID NO 30)	3.6%	26.4%	-0.78
Met-PRT698 (SEQ ID NO 31)	0.0%	26.4%	-0.03
Met-PRT966 (SEQ ID NO 32)	0.0%	28.0%	0.35

The modified fibroin (6-i) may be composed of an amino acid sequence shown in SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 or SEQ ID NO 32.
The modified fibroin (6-ii) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 or SEQ ID NO 32. The modified fibroin (6-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif. The sequence identity is preferably 95% or more.
The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.
The sixth modified fibroin may include a tag sequence at either or both of the N-terminal and the C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
More specific examples of modified fibroins including a tag sequence include (6-iii) a modified fibroin including an amino acid sequence shown in SEQ ID NO 33 (PRT888), SEQ ID NO 34 (PRT965), SEQ ID NO 35 (PRT889), SEQ ID NO 36 (PRT916), SEQ ID NO 37 (PRT918), SEQ ID NO 38 (PRT699), SEQ ID NO 39 (PRT698) or SEQ ID NO 40 (PRT966) or (6-iv) a modified fibroin including an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39 or SEQ ID NO 40.

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

[Table 3]

Modified fibroin	Glutamine residue content	GPGXX motif content	Hydrophobicity of REP
PRT888 (SEQ ID NO 33)	6.3%	27.9%	-0.07
PRT965 (SEQ ID NO 34)	0.0%	27.9%	-0.65
PRT889 (SEQ ID NO 35)	0.0%	27.9%	0.35
PRT916 (SEQ ID NO 36)	0.0%	27.9%	0.47
PRT918 (SEQ ID NO 37)	0.0%	27.9%	0.45
PRT699 (SEQ ID NO 38)	3.6%	26.4%	-0.78
PRT698 (SEQ ID NO 39)	0.0%	26.4%	-0.03
PRT966 (SEQ ID NO 40)	0.0%	28.0%	0.35

The modified fibroin (6-iii) may be composed of an amino acid sequence shown in SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39 or SEQ ID NO 40.
The modified fibroin (6-iv) includes an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39 or SEQ ID NO 40. The modified fibroin (6-iv) is also a protein including a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif. The sequence identity is preferably 95% or more.
The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.
The sixth modified fibroin may include a secretory signal for releasing the protein produced in a recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The modified fibroin may be a modified fibroin having at least two or more characteristics among characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin in combination.
The limit oxygen index (LOI) value of the modified fibroin according to the present embodiment may be 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 29 or more, or 30 or more. In this specification, the LOI value is a value measured according to a test method for powder granules or a synthetic resin having a low melting point, Fire Department Dangerous Goods Regulation .
The maximum hygroscopic heat generation of the modified fibroin according to the present embodiment determined according to the following Formula A may be greater than 0.025°C/g. $Maximum hygroscopic heat generation = \{(maximum value of sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment) - (sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment)\} (° C) / sample weight (g)$
Here, in Formula A, the low humidity environment means an environment of a temperature of 20°C and a relative humidity of 40%, and the high humidity environment means an environment of a temperature of 20°C and a relative humidity of 90%
The maximum hygroscopic heat generation of the modified fibroin according to the present embodiment may be 0.026°C/g or more, 0.027°C/g or more, 0.028°C/g or more, 0.029°C/g or more, 0.030°C/g or more, 0.035°C/g or more, or 0.040°C/g or more. The upper limit of the maximum hygroscopic heat generation is not particularly limited, and is generally 0.060°C/g or less.
The heat retention index of the modified fibroin according to the present embodiment determined according to the following Formula B may be 0.20 or more. $heat retention index = heat retention rate (%) / basis weight (g / m^{2}) of sample$
Here, in this specification, the heat retention rate means a heat retention rate measured by a dry contact method using Thermo labo-II tester (under a wind of 30 cm/sec), and is a value measured by a method described in the section of examples to be descried below.
The heat retention index of the modified fibroin according to the present embodiment may be 0.22 or more, 0.24 or more, 0.26 or more, 0.28 or more, 0.30 or more, or 0.32 or more. The upper limit of the heat retention index is not particularly limited, and may be, for example, 0.60 or less or 0.40 or less.

For example, the modified fibroin according to the present embodiment can be produced by expressing the nucleic acid by a host transformed with an expression vector including a nucleic acid sequence that encodes the modified fibroin and one or more regulatory sequences that are operably linked to the nucleic acid sequence.
A method for manufacturing a nucleic acid that encodes the modified fibroin is not particularly limited. For example, the nucleic acid can be manufactured by a method in which a gene that encodes a natural fibroin is used, amplification and cloning are performed using a polymerase chain reaction (PCR), and modification is performed by a genetic engineering technique or a chemically synthesizing method. A method for chemically synthesizing nucleic acids is not particularly limited, and for example, the gene can be chemically synthesized by a method for linking an oligonucleotide automatically synthesized using AKTA oligopilot plus 10/100 (commercially available from GE Healthcare Japan) through PCR based on amino acid sequence information of fibroins available in the NCBI web database and the like. In this case, in order to facilitate purification and/or confirmation of the modified fibroin, a nucleic acid that encodes a modified fibroin composed of an amino acid sequence obtained by adding an amino acid sequence including a start codon and a His10 tag to the N-terminal of the amino acid sequence may be synthesized.
The regulatory sequence is a sequence (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.) that controls expression of the modified fibroin in the host, and can be appropriately selected depending on the type of the host. Regarding the promoter, an inducible promoter that functions in host cells and can express and induce the modified fibroin may be used. The inducible promoter is a promoter that can control transcription according to the presence of an inducing substance (expression inducer), the absence of repressor molecules, or physical factors such as an increase or decrease in the temperature, the osmotic pressure or the pH value.
The type of the expression vector may be a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector or the like, and can be appropriately selected depending on the type of the host. Regarding the expression vector, a vector which can autonomously replicate in host cells or can be integrated into host chromosomes and contains a promoter at a position at which a nucleic acid that encodes the modified fibroin can be transcripted is suitably used.
Regarding the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungus, insect cells, animal cells and plant cells can be suitably used.
Preferable examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, the genus Brevibacillus, the genus Serratia, the genus Bacillus, the genus Mycobacterium, the genus Corynebacterium, the genus Corynebacterium and the genus Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus Agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganism belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Mycobacterium include Microbacterium ammoniaphilum. Examples of microorganisms belonging to the genus Corynebacterium include Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
When prokaryotes are used as a host, examples of a vector for introducing a nucleic acid that encodes a modified fibroin include pBTrp2 (commercially available from Boehringer Ingelheim GmbH), pGEX (commercially available from Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569 ).
Examples of eukaryotic hosts include yeast and filamentous fungus (mold, etc.). Examples of yeasts include yeasts belonging to the genus Saccharomyces, the genus Pichia, and the genus Schizosaccharomyces. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, and the genus Trichoderma.
When eukaryotes are used as a host, regarding a vector that introduces a nucleic acid that encodes the modified fibroin, for example, YEP13 (ATCC37115) and YEp24 (ATCC37051) may be exemplified. Regarding a method for introducing an 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)], an electroporation method, a spheroplast method, a protoplast method, a lithium acetate method, a competent method, and the like may be exemplified.
Regarding 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, or the like can be performed according to a method described in Molecular Cloning 2^nd edition or the like.
For example, the modified fibroin can be manufactured by culturing a host transformed with an expression vector in a culture medium, generating and storing the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. A method for culturing a host in the culture medium can be performed according to a method that is generally used in culturing of the host.
When the host is prokaryotes such as E. coli or eukaryotes such as yeast, regarding the culture medium, any of a natural culture medium and a synthetic culture medium may be used as long as the culture medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host and in which the host is efficiently cultured.
Regarding the carbon source, any source that can be assimilated by the transformed microorganism may be used, and for example, glucose, fructose, sucrose, and molasses containing them, carbohydrates such as starch and starch hydrolyzate, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used. Regarding the nitrogen source, for example, inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or ammonium salts of organic acids, other nitrogen-containing compounds, and peptone, meat extracts, yeast extracts, corn steep liquors, casein hydrolyzates, soybean cakes and soybean cake hydrolyzates, various fermented bacteria and their digested products can be used. Regarding inorganic salts, for example, potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
Prokaryotes such as E. coli or eukaryotes such as yeast can be cultured, for example, under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40°C. The culture time is generally 16 hours to 7 days. The pH of a culture medium during culturing is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, and the like.
In addition, during culturing, as necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium. When microorganisms transformed with an expression vector using an inducible promoter as a promoter are cultured, as necessary, an inducer may be added to the culture medium. For example, when microorganisms transformed with an expression vector using a lac promoter are cultured, isopropyl-β-D-thiogalactopyranoside or the like may be added to the culture medium, and when microorganisms transformed with an expression vector using a trp promoter are cultured, indole acrylic acid or the like may be added to the culture medium.
The expressed modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a dissolved state in cells, after culturing, host cells are collected by centrifugation, and suspended in an aqueous buffer solution, and the host cells are then crushed with an ultrasonic crusher, a French press, a Manton-Gaulin homogenizer, Dyno Mill or the like, and thereby a cell-free extract liquid is obtained. Methods commonly used for isolating and purifying the modified fibroin from a supernatant obtained by centrifuging the cell-free extract liquid, that is, methods such as a solvent extraction method, a salting-out method using ammonium sulfate or the like, a desalting method, a precipitation method using an organic solvent, an anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-sepharose and DIAION HPA-75 (commercially available from Mitsubishi Chemical Corporation), a cation exchange chromatography method using a resin such as S-Sepharose FF (commercially available from Pharmacia), a hydrophobic chromatography method using a resin such as butyl sepharose and phenyl sepharose, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, and an electrophoresis method such as an isoelectric point electrophoresis method may be used alone or in combination, and thereby a purified preparation can be obtained.
In addition, when the modified fibroin is expressed by forming an insoluble component in cells, host cells are collected in the same manner, and then crushed and centrifuged, and thus insoluble components of the modified fibroin are collected as a precipitate fraction. The collected insoluble components of the modified fibroin can be solubilized with a protein denaturation agent. After the operation, a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be collected from a culture supernatant. That is, the culture product is treated with a technique such as centrifugation to obtain a culture supernatant, and a purified preparation can be obtained from the culture supernatant using the same isolation and purification method as described above.

The modified fibroin fiber (filament) according to the present embodiment is obtained by spinning the above modified fibroin, and includes the above modified fibroin as a main component. The modified fibroin fiber can be manufactured by a known spinning method. That is, for example, when a modified fibroin fiber including a modified fibroin as a main component is manufactured, first, the modified fibroin manufactured according to the above method is added to a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP), and as necessary, an inorganic salt such as a dissolution promoting agent is added, and dissolved to prepare a dope solution. Then, using this dope solution, spinning is performed by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning, and thereby a modified fibroin fiber can be obtained. Examples of preferable spinning methods include a wet spinning method and a dry wet spinning method.
FIG. 6 is an explanatory diagram schematically showing an example of a spinning device for manufacturing a modified fibroin fiber. A spinning device 10 shown in FIG. 6 is an example of a spinning device for dry wet spinning, and includes an extrusion device 1, an unstretched yarn manufacturing device 2, a wet heat stretching device 3, and a drying device 4.
A spinning method using the spinning device 10 will be described. First, a dope solution 6 stored in a storage tank 7 is extruded from a cap 9 by a gear pump 8. In the laboratory scale, the dope solution may be filled in a cylinder and extruded from a nozzle using a syringe pump. Then, the extruded dope solution 6 is supplied to a coagulating liquid 11 in a coagulating liquid tank 20 through an air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulated component is formed. Next, the fibrous coagulated component is supplied to hot water 12 in a stretched bathtub 21 and stretched. The draw ratio is determined by a speed ratio between a supply nip roller 13 and a take-off nip roller 14. Then, the stretched fibrous coagulated component is supplied to the drying device 4 and dried in a yarn passage 22, and a modified fibroin fiber 36 is obtained as a yarn wound component 5. 18a to 18g are yarn guides.
The coagulating liquid 11 may be any solution in which a solvent can be removed, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulating liquid 11 may appropriately contain water. The temperature of the coagulating liquid 11 is preferably 0 to 30°C. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the cap 9, the extrusion rate per hole is preferably 0.2 to 6.0 ml/hr and more preferably 1.4 to 4.0 ml/hr. The distance that the coagulated modified fibroin passes through the coagulating liquid 11 (substantially, a distance from the yarn guide 18a to the yarn guide 18b) may be any length at which the solvent can be efficiently removed, and is, for example, 200 to 500 mm. The take-up speed of the unstretched yarn may be, for example, 1 to 20 m/min, and is preferably 1 to 3 m/min. A time for which the yarn is retained in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and is preferably 0.05 to 0.15 minutes. In addition, stretching (pre-stretching) may be performed in the coagulating liquid 11. The coagulating liquid tank 20 may be provided in multiple stages, and stretching may be performed in each stage or a specific stage as necessary.
Here, regarding the stretching performed when the modified fibroin fiber is obtained, for example, in addition to pre-stretching performed in the coagulating liquid tank 20 and wet heat stretching performed in the stretched bathtub 21 described above, dry heat stretching is also used.
The wet heat stretching can be performed in hot water, in a solution obtained by adding an organic solvent and the like to hot water, or during steam heating. The temperature may be, for example, 50 to 90°C, and is preferably 75 to 85°C. In the wet heat stretching, for example, an unstretched yarn (or pre-stretched yarn) can be stretched 1 to 10 times, and preferably stretched 2 to 8 times.
Dry heat stretching can be performed using an electric tubular furnace, a dry hot plate, or the like. The temperature may be, for example, 140°C to 270°C, and is preferably 160°C to 230°C. In the dry heat stretching, the unstretched yarn (or pre-stretched yarn) can be stretched, for example, 0.5 to 8 times, and preferably stretched 1 to 4 times.
The wet heat stretching and the dry heat stretching may be performed alone or in multiple stages, or in combination. That is, the wet heat stretching and the dry heat stretching can be appropriately performed in combination, for example, first stage stretching is performed by wet heat stretching and second stage stretching is performed by dry heat stretching or first stage stretching is performed by wet heat stretching, second stage stretching is performed by wet heat stretching, and additionally third stage stretching is performed by dry heat stretching.
The lower limit value of the final draw ratio is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, or 9 times or more that of the unstretched yarn (or pre-stretched yarn), and the upper limit value 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, or 10 times or less that of the unstretched yarn (or pre-stretched yarn).

An artificial fibroin spun yarn can be obtained by a method including a cutting process in which the above modified fibroin fiber (filament) is cut into an appropriate length to obtain a modified fibroin staple and a spinning process in which the obtained modified fibroin staple is spun.
The cutting process can be performed using any device that can cut the modified fibroin fiber. Examples of such a device include a desktop fiber cutting machine (s/NO. IT-160201-NP-300 ).
The length of the modified fibroin staple is not particularly limited, and may be, for example, 20 mm or more, 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.
The spinning process can be performed by a known spinning method. Examples of spinning methods include a cotton spinning method, a worsted spinning method and a woolen spinning method. A device used for these spinning methods is not particularly limited, and a commonly used device can be used. In addition, in the spinning process, first, the modified fibroin staple may be opened or defibrated by a fiber-opening machine (opener), a defibrating machine (breaker) or the like.
The method for manufacturing an artificial fibroin spun yarn may include a crimping process before or after the cutting process. The crimping process may be, for example, a process in which a mechanical crimping method such as an indentation method is performed or may be a process in which a modified fibroin fiber (filament) or a modified fibroin staple is brought into contact with an aqueous medium and crimped (hereinafter referred to as "water crimp" in some cases).
The aqueous medium is a liquid medium containing water (including water vapor) or a gas (steam) medium. The aqueous medium may be water or a liquid in which water and a hydrophilic solvent are mixed. In addition, regarding the hydrophilic solvent, for example, a volatile solvent such as ethanol and methanol or vapors thereof can be used. The aqueous medium may be a liquid in which water and a volatile solvent such as ethanol and methanol are mixed and is preferably water or a liquid in which water and ethanol are mixed. When an aqueous medium containing a volatile solvent or vapors thereof is used, it is possible to increase a drying speed after water crimping, and additionally, it is possible to impart soft texture to the finally obtained crimped staple. The ratio between water and the volatile solvent or vapors thereof is not particularly limited, and for example, the mass ratio of water:volatile solvent or vapors thereof may be 10:90 to 90:10. The ratio of water is preferably 30 mass% or more, and may be 40 mass% or 50 mass% or more.
The aqueous medium is preferably a liquid or gas containing water (including water vapor) at 10 to 230°C. The temperature of the aqueous medium may be 10°C or higher, 25°C or higher, 40°C or higher, 60°C or higher, or 100°C or higher, and may be 230°C or lower, 120°C or lower, or 100°C or lower. More specifically, when the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably 100 to 230°C, and more preferably 100 to 120°C. When the steam of the aqueous medium is at 230°C or lower, it is possible to prevent denaturation of the modified fibroin filament by heat. When the aqueous medium is a liquid, the temperature of the aqueous medium is preferably 10°C or higher, 25°C or higher, or 40°C or higher in order to efficiently impart crimp, and is preferably 60°C or lower in order to keep the fiber strength of the modified fibroin filament high.
A time for which the aqueous medium is in contact is not particularly limited, and may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer, and is preferably 10 minutes or shorter in consideration of the productivity. In addition, in the case of steam, it is considered that a large shrinkage rate is obtained in a shorter time than in the case of a liquid. The contact with the aqueous medium may be performed under an atmospheric pressure or under a reduced pressure (for example, vacuum).
Examples of a method for contacting with an aqueous medium include a method for immersing a modified fibroin filament or a modified fibroin staple in an aqueous medium, a method for spraying aqueous medium steam to a modified fibroin filament or a modified fibroin staple, and a method for exposing a modified fibroin filament or a modified fibroin staple to an environment filled with aqueous medium steam. When the aqueous medium is steam, the contact of the aqueous medium with the modified fibroin filament or the modified fibroin staple can be performed using a general steam setting device. Specific examples of steam setting devices include devices such as product name: FMSA type steam setter-(commercially available from Fukushin Kougyo Co., Ltd.), and product name: EPS-400 (commercially available from Tsujii Dyeing Machine Manufacturing Co., Ltd.). Specific examples of a method for crimping a modified fibroin filament or a modified fibroin staple with aqueous medium steam include a method in which a modified fibroin filament or a modified fibroin staple is accommodated in a predetermined accommodating chamber, aqueous medium steam is introduced into the accommodating chamber, the temperature in the accommodating chamber is adjusted to the above predetermined temperature (for example, 100°C to 230°C), and steam is brought into contact with the modified fibroin filament or the modified fibroin staple.
Here, the process of crimping the modified fibroin filament or the modified fibroin staple by contact with an aqueous medium is performed preferably in a state in which no tensile force is applied to single fibers and bundles (hanks, etc.) of the modified fibroin filament and the modified fibroin staple (no tension is applied in the fiber axis direction) or in a state in which only a predetermined degree is applied (a predetermined amount of tension is applied in the fiber axis direction). When a tensile force applied to the modified fibroin filament or the modified fibroin staple in this case is adjusted, it is possible to control the degree of crimping. Examples of a method for preparing a tensile force applied to the modified fibroin filament and the modified fibroin staple include a method for adjusting a load applied to the filament and the staple by hanging weights with various weights on the modified fibroin filament and the modified fibroin staple, a method for fixing both ends while the filament and the staple are slackened and changing the amount of slackness variously, and a method for winding the filament around a winding target object such as a paper tube or a bobbin, and appropriately changing a winding force (a tightening force on the paper tube or the bobbin) in this case.
The modified fibroin filament or the modified fibroin staple is brought into contact with the aqueous medium and may be then additionally dried. The drying method is not particularly limited, and the drying may be performed by natural drying, or the drying may be performed with hot air or a hot roller. The drying temperature is not particularly limited, and may be for example, 20 to 150°C, and is preferably 40 to 120°C, and more preferably 60 to 100°C.
Here, in order to further increase the shrinkage rate of the artificial fibroin spun yarn, it is preferable not to perform the crimping process (particularly a water crimping process). In addition, even if the crimping process is performed, when spinning is performed using a staple in which the crimped staple and the uncrimped staple are mixed in the spinning process, it is possible to further increase the shrinkage rate of the artificial fibroin spun yarn.
The artificial fibroin spun yarn may be a single yarn or a mixed yarn such as a twin yarn. Examples of artificial fibroin spun yarns include a spun yarn composed of 100% modified fibroin, a mixed yarn composed of a staple of 100% modified fibroin and at least one selected from among other protein staples, chemical fiber staples and the like, a spun yarn using a staple containing a modified fibroin and other components, and a mixed yarn composed of a staple containing a modified fibroin and other components and at least one selected from among other protein staples and chemical fiber staples.
In order to make the modified fibroin staple easily loosen, an oil agent may be attached in advance before the spinning process. The oil agent can be attached in any stage in the manufacturing process. For example, the oil agent may be attached before the cutting process, at the same time as the cutting process, or after the cutting process. The oil agent is not particularly limited, and any of known oil agents that are used for general purposes for imparting processability and functionality such as for imparting an antistatic property, for reducing friction, for imparting flexibility, or imparting water repellency can be used.

An artificial fibroin spun yarn according to the present embodiment includes the above modified fibroin and is highly shrinkable when it is simply in contact with water (water, water vapor, etc.). The term "highly shrinkable" means that the shrinkage rate defined in the following Formula II is more than 1%. $Shrinkage rate = \{1 - (length of artificial fibroin spun yarn subjected to shrinkage processing including being brought into contact with water having a temperature lower than a boiling point / length of artificial fibroin spun yarn before the above shrinkage processing is performed)\} \times 100 (%)$
The artificial fibroin spun yarn according to the present embodiment includes the above modified fibroin, and has a shrinkage rate of greater than 1% defined in Formula II, and thus can shrink at a higher shrinkage rate than protein spun yarns known in the related art, and can shrink at a higher shrinkage rate under moderate conditions than spun yarns composed of synthetic fibers known in the related art.
The shrinkage rate of the artificial fibroin spun yarn according to the present embodiment may be 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more. The upper limit of the shrinkage rate is not particularly limited, and may be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less.
In addition, the artificial fibroin spun yarn according to the present embodiment includes the modified fibroin fiber (which means a filament after spinning) as a raw material, and is highly shrinkable when there is no history of contact with water (water, water vapor, etc.). That is, when there is no history of contact with water, the shrinkage rate of the artificial fibroin spun yarn according to the present embodiment may be, for example, more than 7%, and is preferably 15% or more, more preferably more than 25%, still more preferably 32% or more, still more preferably 40% or more, still more preferably 48% or more, particularly preferably 56% or more, more particularly preferably 64% or more, and most preferably 72% or more. The upper limit of the shrinkage rate is generally 80% or less.
The limit oxygen index (LOI) value of the artificial fibroin spun yarn according to the present embodiment may be 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 29 or more, or 30 or more.
The artificial fibroin spun yarn according to the present embodiment has a maximum hygroscopic heat generation of greater than 0.025°C/g determined according to the following Formula A. $Maximum hygroscopic heat generation = \{(maximum value of sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment) - (sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment)\} (° C) / sample weight (g)$
Here, in Formula A, the low humidity environment means an environment of a temperature of 20°C and a relative humidity of 40%, and the high humidity environment means an environment of a temperature of 20°C and a relative humidity of 90%.
The maximum hygroscopic heat generation of the artificial fibroin spun yarn according to the present embodiment may be 0.026°C/g or more, 0.027°C/g or more, 0.028°C/g or more, 0.029°C/g or more, 0.030°C/g or more, 0.035°C/g or more, or 0.040°C/g or more. The upper limit of the maximum hygroscopic heat generation is not particularly limited, and is generally 0.060°C/g or less.
The heat retention index of the artificial fibroin spun yarn according to the present embodiment determined according to the following Formula B may be 0.20 or more. $heat retention index = heat retention rate (%) / basis weight (g / m^{2}) of sample$
The heat retention index of the artificial fibroin spun yarn according to the present embodiment may be 0.22 or more, 0.24 or more, 0.26 or more, 0.28 or more, 0.30 or more, or 0.32 or more. The upper limit of the heat retention index is not particularly limited, and may be, for example, 0.60 or less or 0.40 or less.

(Shrinkage processing)

The shrinkage processing includes bringing an artificial fibroin spun yarn into contact with water having a temperature lower than a boiling point (hereinafter referred to as a "contact step"). The shrinkage processing may further include drying of the artificial fibroin spun yarn after contact with water (after the contact step) (hereinafter referred to as a "drying step").
The temperature of water in contact with the artificial fibroin spun yarn in the contact step may be lower than the boiling point. Thereby, handling properties and workability of shrinkage processing and the like are improved. In addition, in order to sufficiently shorten the shrinking time, the lower limit value of the temperature of water is preferably 10°C or higher, more preferably 40°C or higher, and still more preferably 70°C or higher. The upper limit value of the temperature of water is preferably 90°C or lower.
In the contact step, a method for bringing water into contact with the artificial fibroin spun yarn is not particularly limited. Examples of the method include a method for immersing an artificial fibroin spun yarn in water, a method for spraying water at room temperature or in a heated state such as steam to an artificial fibroin spun yarn, and a method for exposing an artificial fibroin spun yarn to a high humidity environment filled with water vapor. Among these methods, in the contact step, a method for immersing an artificial fibroin spun yarn in water is preferable because the shrinking time can be effectively shortened and processing facilities can be simplified.
The drying step is a step of drying the artificial fibroin spun yarn that has undergone the contact step. For example, the drying may be performed by natural drying or may be forcibly performed by using a drying device. Regarding the drying device, any known contact type or non-contact type drying device can be used. In addition, the drying temperature is not particularly limited as long as the temperature is, for example, lower than a temperature at which proteins (modified fibroin, etc.) contained in the artificial fibroin spun yarn are decompose and the artificial fibroin spun yarn is thermally damaged, and is generally a temperature in a range of 20 to 150°C and preferably a temperature in a range of 50 to 100°C. When the temperature is in this range, the artificial fibroin spun yarn is more quickly and efficiently dried without causing thermal damage to the artificial fibroin spun yarn or decomposition of proteins contained in the artificial fibroin spun yarn. The drying time is appropriately set according to the drying temperature and the like, and for example, a time at which the influence of overdrying on the quality, physical properties, and the like of the artificial fibroin spun yarn can be eliminated as much as possible is used.
The shrinkage rate of the artificial fibroin spun yarn according to the present embodiment can be controlled by controlling, for example, the temperature of water in contact, the contact time with water, and the tensile force when brought into contact with water.

<High-shrinkage artificial fibroin spun yarn and method for manufacturing the same>

Since the artificial fibroin spun yarn according to the present embodiment is highly shrinkable, for example, the high-shrinkage artificial fibroin spun yarn can be obtained through the above shrinkage processing (the contact step, and as necessary the drying step).
In the high-shrinkage artificial fibroin spun yarn according to the present embodiment, the shrinkage rate defined by the following Formula I is more than 1%. $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$
The shrinkage rate of the high-shrinkage artificial fibroin spun yarn according to the present embodiment may be 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more. The upper limit of the shrinkage rate is not particularly limited, and may be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less.
In addition, the artificial fibroin spun yarn according to the present embodiment includes the modified fibroin fiber (which means a filament after spinning) as a raw material, and is highly shrinkable when there is no history of contact with water (water, water vapor, etc.). That is, when there is no history of contact with water, the shrinkage rate of the high-shrinkage artificial fibroin spun yarn according to the present embodiment may be, for example, more than 7%, and is preferably 15% or more, more preferably more than 25%, still more preferably 32% or more, still more preferably 40% or more, still more preferably 48% or more, particularly preferably 56% or more, more particularly preferably 64% or more, and most preferably 72% or more. The upper limit of the shrinkage rate is generally 80% or less.
The limit oxygen index (LOI) value of the artificial fibroin spun yarn according to the present embodiment may be 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 29 or more, or 30 or more.
The maximum hygroscopic heat generation of the artificial fibroin spun yarn according to the present embodiment determined according to the following Formula A may be greater than 0.025°C/g. $Maximum hygroscopic heat generation = \{(maximum value of sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment) - (sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment)\} (° C) / sample weight (g)$
Here, in Formula A, the low humidity environment means an environment of a temperature of 20°C and a relative humidity of 40%, and the high humidity environment means an environment of a temperature of 20°C and a relative humidity of 90%.
The maximum hygroscopic heat generation of the artificial fibroin spun yarn according to the present embodiment may be 0.026°C/g or more, 0.027°C/g or more, 0.028°C/g or more, 0.029°C/g or more, 0.030°C/g or more, 0.035°C/g or more, or 0.040°C/g or more. The upper limit of the maximum hygroscopic heat generation is not particularly limited, and is generally 0.060°C/g or less.
The heat retention index of the artificial fibroin spun yarn according to the present embodiment determined according to the following Formula B may be 0.20 or more. $heat retention index = heat retention rate (%) / basis weight (g / m^{2}) of sample$
The heat retention index of the artificial fibroin spun yarn according to the present embodiment may be 0.22 or more, 0.24 or more, 0.26 or more, 0.28 or more, 0.30 or more, or 0.32 or more. The upper limit of the heat retention index is not particularly limited, and may be, for example, 0.60 or less or 0.40 or less.

[Method for shrinking artificial fibroin spun yarn]

A method for shrinking an artificial fibroin spun yarn according to the present embodiment includes a process of bringing an artificial fibroin spun yarn including a modified fibroin into contact with water having a temperature lower than a boiling point and performing shrinking. The shrinkage process may further include drying of the artificial fibroin spun yarn after contact with water. That is, in the same manner as in the above shrinkage processing, the method for shrinking an artificial fibroin spun yarn according to the present embodiment can be performed.

Examples

The present invention will be described below in more detail with reference to examples and the like. However, the present invention is not limited to the following examples.

[Test Example 1: manufacture of modified fibroin (modified spider silk fibroin)]

(1) Production of modified fibroin expression vector

A modified fibroin (PRT399) having an amino acid sequence shown in SEQ ID NO 18, a modified fibroin (PRT380) having an amino acid sequence shown in SEQ ID NO 12, a modified fibroin (PRT410) having an amino acid sequence shown in SEQ ID NO 13, a modified fibroin (PRT799) having an amino acid sequence shown in SEQ ID NO 15, and a modified fibroin (PRT918) having an amino acid sequence shown in SEQ ID NO 37 were designed.
Nucleic acids that encode a protein having the designed amino acid sequence were synthesized. In the nucleic acids, an NdeI site was added to the 5' end and an EcoRI site was added downstream of the stop codon. These five nucleic acids were cloned into cloning vectors (pUC118). Then, the same nucleic acid was treated with restriction enzymes NdeI and EcoRI and cut, and then recombined into a protein expression vector pET-22b(+) to obtain an expression vector.

(2) Expression of modified fibroin

E. coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in a 2 mL LB culture medium containing ampicillin for 15 hours. The culture solution was added to a 100 mL culture medium containing ampicillin for seed culturing (Table 4) so that the OD₆₀₀ became 0.005. The temperature of the culture solution was maintained at 30°C and flask culture was performed until the OD₆₀₀ became 5 (about 15 hours), and thereby a seed culture solution was obtained. [Table 4]

Culture medium for seed culturing

Sample Concentration (g/L)

Glucose 5.0

KH₂PO₄ 4.0

K₂HPO₄ 9.3

Yeast Extract 6.0

Ampicillin 0.1

The seed culture solution was added to a jar fermenter containing a 500 ml production culture medium (Table 5) so that the OD₆₀₀ became 0.05. The temperature of the culture solution was maintained at 37°C and the culture was performed under control with a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved saturated oxygen concentration.

[Table 5]

Production culture medium
Sample	Concentration (g/L)
Glucose	12.0
KH₂PO₄	9.0
MgSO₄·7H₂O	2.4
Yeast Extract	15
FeSO₄·7H₂O	0.04
MnSO₄·5H₂O	0.04
CaCl₂·2H₂O	0.04
ADEKA NOL (ADEKA, LG-295S)	0.1 (mL/L)

Immediately after glucose in the production culture 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 temperature of the culture solution was maintained at 37°C and the culture was performed under control with a constant pH of 6.9. The culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved saturated oxygen concentration. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution so that the final concentration was 1 mM, and thereby a desired modified fibroin was expressed and induced. When 20 hours had elapsed after IPTG was added, the culture solution was centrifuged, and bacteria were collected. SDS-PAGE was performed using bacteria prepared from the culture solution before IPTG was added and after IPTG was added, and the expression of the desired modified fibroin was confirmed according to the appearance of a band with a size corresponding to the desired modified fibroin depending on the addition of IPTG.

(3) Purification of modified fibroin

The bacteria collected 2 hours after IPTG was added were washed with a 20 mM Tris-HCl buffer (pH 7.4). The bacteria after washing were suspended in a 20 mM Tris-HCl buffer solution (pH 7.4) containing about 1 mM PMSF, and cells were crushed using a high pressure homogenizer (commercially available from GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer solution (pH 7.4) until it became highly pure. The precipitate after washing was suspended in a 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so that the concentration became 100 mg/mL, and stirred with a stirrer at 60°C for 30 minutes and dissolved. After the dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 commercially available from Sanko Junyaku Co., Ltd.). White aggregated proteins obtained after dialysis were collected through centrifugation. Water was removed from the collected aggregated proteins in a freeze dryer, and thereby lyophilized powders of desired modified fibroins (PRT399, PRT380, PRT410, PRT799 and PRT918) were obtained.

[Reference Example 1: manufacture of modified fibroin fiber (filament) and evaluation of shrinkability]

Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass% was prepared as a solvent, and the lyophilized powder of the modified fibroin (PRT399, PRT380, PRT410 or PRT799) obtained in Test Example 1 was added thereto at a concentration of 18 mass% or 24 mass% (refer to Table 6), and dissolved using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to obtain a modified fibroin solution.
The obtained modified fibroin solution was used as a dope solution (spinning stock solution), and a spun and stretched modified fibroin fiber (filament) was manufactured by dry wet spinning using a spinning device similar to the spinning device 10 shown in FIG. 6. The spinning device that was used was obtained by additionally providing a second unstretched yarn manufacturing device (a second bath) between the unstretched yarn manufacturing device 2 (a first bath) and the wet heat stretching device 3 (a third bath) in the spinning device 10 shown in FIG. 6. The dry wet spinning conditions were as follows.

Extrusion nozzle diameter: 0.2 mm

Liquids and temperatures in the first bath to the third bath: refer to Table 6
Total draw ratio: refer to Table 6
Drying temperature: 60°C

[Table 6]

	Dope solution		First bath		Second bath		Third bath		Total draw ratio (times)
	Modified Concentration fibroin (mass%)		Liquid	Temperature (°C)	Liquid Temperature (°C)		Liquid	Temperature (°C)	Total draw ratio (times)
Manufacturing Example 1	PRT799	24	100% methanol	-5	100% methanol	16	Water	17	1
Manufacturing Example 2									2
Manufacturing Example 3									3
Manufacturing Example 4									4
Manufacturing Example 5		18							1
Manufacturing Example 6									2
Manufacturing Example 7									3
Manufacturing Example 8									4
Manufacturing Example 9	PRT410	24		-11		14			1
Manufacturing Example 10									2
Manufacturing Example 11									3
Manufacturing Example 12									4
Manufacturing Example 13	PRT399								1
Manufacturing Example 14									2
Manufacturing Example 15									3
Manufacturing Example 16	PRT380					11			1
Manufacturing Example 17									2
Manufacturing Example 18									3
Manufacturing Example 19									4

(Evaluation of shrinkability)

The shrinkage rates of the modified fibroin fibers (filaments) obtained in Manufacturing Examples 1 to 19 were evaluated. That is, regarding the modified fibroin fibers, a shrinkage rate when shrinkage processing 1 including being brought into contact with water having a temperature lower than a boiling point was performed (hereinafter referred to as a "primary shrinkage rate"), and a shrinkage rate when shrinkage processing 2 including performing drying at room temperature after bringing into contact with water having a temperature lower than a boiling point were performed (hereinafter referred to as a "secondary shrinkage rate") were evaluated.

A plurality of modified fibroin fibers for testing, each having a length of 30 cm, were cut out from a wound product of the modified fibroin fibers obtained in Manufacturing Examples 1 to 19. The plurality of modified fibroin fibers were bundled to obtain a modified fibroin fiber bundle having a fineness of 150 denier. A 0.8 g plumb bob was attached to each modified fibroin fiber bundle, and in that state, each modified fibroin fiber bundle was immersed in water at a temperature shown in Tables 7 to 10 for 10 minutes (the shrinkage processing 1). Then, the length of each modified fibroin fiber bundle was measured in water. The length of the modified fibroin fiber bundle in water was measured while the 0.8 g plumb bob was attached to the modified fibroin fiber bundle in order to eliminate curling of the modified fibroin fiber bundle. Then, the primary shrinkage rate (%) of each modified fibroin fiber was calculated according to the following Formula III. In Formula III, L0 represents the length (here, 30 cm) of the modified fibroin fiber bundle before shrinkage processing was performed, and Lw represents the length of the modified fibroin fiber bundle subjected to the shrinkage processing 1. $shrinkage rate (primary shrinkage rate) = \{1 - (Lw / L 0)\} \times 100 (%)$

After immersion in water for evaluation of the primary shrinkage rate, the modified fibroin fiber bundle was removed from water. The removed modified fibroin fiber bundle with the 0.8 g plumb bob attached thereto was dried at room temperature for 2 hours (the shrinkage processing 2). After the drying, the length of each modified fibroin fiber bundle was measured. Then, the secondary shrinkage rate (%) of each modified fibroin fiber was calculated according to the following Formula IV. In Formula IV, L0 represents the length (here, 30 cm) of the modified fibroin fiber bundle before shrinkage processing was performed, and Lwd represents the length of the modified fibroin fiber bundle subjected to the shrinkage processing 2. $shrinkage rate (secondary shrinkage rate) = \{1 - (Lwd / L 0)\} \times 100 (%)$

The results are shown in Tables 7 to 10.

[Table 7]

Modified fibroin fiber		Temperature of water lower than boiling point (°C)	Primary shrinkage rate (%)	Secondary shrinkage rate (%)
Manufacturing Example 1	24 wt% PRT799 x1	20	0.0	7.8
Manufacturing Example 2	24 wt% PRT799 x2		-1.2	10.3
Manufacturing Example 3	24 wt% PRT799 x3		7.2	21.2
Manufacturing Example 4	24 wt% PRT799 x4		13.5	26.3
Manufacturing Example 6	18 wt% PRT799 x2		-2.3	9.5
Manufacturing Example 7	18 wt% PRT799 x3		6.0	19.7
Manufacturing Example 8	18 wt% PRT799 x4		14.3	27.5
Manufacturing Example 2	24 wt% PRT799 x2	40	-5.3	7.2
Manufacturing Example 3	24 wt% PRT799 x3		8.7	21.3
Manufacturing Example 4	24 wt% PRT799 x4		14.5	26.0
Manufacturing Example 6	18 wt% PRT799 x2		-4.3	7.3
Manufacturing Example 7	18 wt% PRT799 x3		6.2	18.3
Manufacturing Example 8	18 wt% PRT799 x4		16.0	28.7
Manufacturing Example 3	24 wt% PRT799 x3	60	6.8	21.0
Manufacturing Example 4	24 wt% PRT799 x4		15.0	27.5
Manufacturing Example 6	18 wt% PRT799 x2		-1.5	10.7
Manufacturing Example 7	18 wt% PRT799 x3		3.3	18.2
Manufacturing Example 8	18 wt% PRT799 x4		16.2	29.0

[Table 8]

Modified fibroin fiber		Temperature of water lower than boiling point (°C)	Primary shrinkage rate (%)	Secondary shrinkage rate (%)
Manufacturing Example 10	24 wt% PRT410 x2	20	-2.3	8.7
Manufacturing Example 11	24 wt% PRT410 x3		4.7	16.7
Manufacturing Example 12	24 wt% PRT410 x4		10.3	22.3
Manufacturing Example 11	24 wt% PRT410 x3	40	4.7	17.5
Manufacturing Example 12	24 wt% PRT410 x4	40	11.5	24.0
Manufacturing Example 11	24 wt% PRT410 x3	60	2.0	16.5
Manufacturing Example 12	24 wt% PRT410 x4	60	10.8	25.0

[Table 9]

Modified fibroin fiber		Temperature of water lower than boiling point (°C)	Primary shrinkage rate (%)	Secondary shrinkage rate (%)
Manufacturing Example 13	24 wt% PRT399 x1	20	-3.5	7.6
Manufacturing Example 14	24 wt% PRT399 x2		3.7	12.5
Manufacturing Example 15	24 wt% PRT399 x3		7.0	16.8
Manufacturing Example 14	24 wt% PRT399 x2	40	3.0	12.7
Manufacturing Example 15	24 wt% PRT399 x3	40	7.3	16.7
Manufacturing Example 14	24 wt% PRT399 x2	60	3.3	9.3
Manufacturing Example 15	24 wt% PRT399 x3	60	6.8	14.2

[Table 10]

Modified fibroin fiber		Temperature of water lower than boiling point (°C)	Primary shrinkage rate (%)	Secondary shrinkage rate (%)
Manufacturing Example 16	24 wt% PRT380 x1	20	-1.1	9.4
Manufacturing Example 17	24 wt% PRT380 x2		2.7	13.3
Manufacturing Example 18	24 wt% PRT380 x3		7.0	17.7
Manufacturing Example 19	24 wt% PRT380 x4		10.0	20.2
Manufacturing Example 17	24 wt% PRT380 x2	40	3.3	14.2
Manufacturing Example 18	24 wt% PRT380 x3		7.7	19.0
Manufacturing Example 19	24 wt% PRT380 x4		12.0	22.0
Manufacturing Example 17	24 wt% PRT380 x2	60	2.7	14.3
Manufacturing Example 18	24 wt% PRT380 x3		8.2	20.3
Manufacturing Example 19	24 wt% PRT380 x4		12.0	23.2

Based on the above results, it was found that the modified fibroin fiber (filament) according to the present invention had a sufficiently high shrinkage rate. Therefore, it was confirmed that the artificial fibroin spun yarn spun from the modified fibroin fiber (filament) according to the present invention had a sufficiently high shrinkage rate. In addition, it can be understood that the shrinkage rate of the modified fibroin fiber (filament) and the artificial fibroin spun yarn according to the present invention can be controlled by controlling, for example, the temperature of water in contact, the contact time with water, and the tensile force when brought into contact with water.

[Example 1: manufacture and evaluation of artificial fibroin spun yarn]

(1) Manufacture of modified fibroin fiber (filament)

Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass% was prepared as a solvent, and the lyophilized powder of the modified fibroin (PRT799) obtained in Test Example 1 was added thereto at a concentration of 24 mass%, and dissolved using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to obtain a modified fibroin solution.
The obtained modified fibroin solution was used as a dope solution (spinning stock solution), and dry wet spinning was performed using a spinning device similar to the spinning device 10 shown in FIG. 6 to obtain modified fibroin filaments (24 multifilaments). The obtained modified fibroin filaments were wound on a bobbin. The dry wet spinning conditions were as follows.
Temperature of coagulating liquid (methanol): 5 to 10°C
Draw ratio: 5 times
Drying temperature: 80°C

(2) Manufacture of artificial fibroin spun yarn

The modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm using a desktop fiber cutting machine to obtain a modified fibroin staple. A part of the obtained staple was immersed in water at 40°C for 1 minute and curled and crimped and then dried at 40°C for 18 hours to obtain a crimped staple. Then, a staple including the crimped staple and the uncrimped staple at a ratio of 7:3 (weight ratio) was spun using a known spinning device to obtain an artificial fibroin spun yarn 1. The fineness (yarn number) of the artificial fibroin spun yarn 1 was 30 Nm.

(3) Evaluation of artificial fibroin spun yarn (water shrinkage test)

The artificial fibroin spun yarn 1 was cut into a length of 60 cm and immersed in water at 40°C for 1 minute and then dried with air. The length of the spun yarn dried with air was measured, and the shrinkage rate was determined according to the following Formula V. The results are shown in Table 11. $Shrinkage rate = \{1 - (length of spun yarn dried with air / length of spun yarn before immersion in water)\} \times 100 (%)$
Here, the "length of the spun yarn before immersion in water" was 60 cm.

[Example 2: manufacture and evaluation of artificial fibroin spun]

(1) Manufacture of modified fibroin fiber (filament)

A modified fibroin filament was obtained in the same method as in Example 1.

(2) Manufacture of artificial fibroin spun yarn

The modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm using a desktop fiber cutting machine to obtain a modified fibroin staple. The obtained staple (uncrimped staple) was spun using a known spinning device to obtain an artificial fibroin spun yarn 2. The fineness (yarn number) of the artificial fibroin spun yarn 2 was 48 Nm.

(3) Evaluation of artificial fibroin spun yarn (water shrinkage test)

The shrinkage rate of the artificial fibroin spun yarn 2 was determined in the same method as in Example 1. The results are shown in Table 11.

[Comparative Example 1: manufacture and evaluation of artificial fibroin spun yarn]

(1) Manufacture of modified fibroin fiber (filament)

A modified fibroin filament was manufactured in the same manner as in Example 1 except that, in place of lyophilized powder of the modified fibroin (PRT799), lyophilized powder in which the lyophilized powder of the modified fibroin (PRT918) and the lyophilized powder of the modified fibroin (PRT799) obtained in Test Example 1 were mixed at a ratio of 6:4 (weight ratio) was used.

(2) Manufacture of artificial fibroin spun yarn

The modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm using a desktop fiber cutting machine to obtain a modified fibroin staple. The obtained staple was immersed in water at 40°C for 1 minute and curled and crimped, and then dried at 40°C for 18 hours to obtain a crimped staple. Then, the crimped staple was spun using a known spinning device to obtain an artificial fibroin spun yarn 3. The fineness (yarn number) of the artificial fibroin spun yarn 3 was 3 Nm.

(3) Evaluation of artificial fibroin spun yarn (water shrinkage test)

The shrinkage rate of the artificial fibroin spun yarn 3 was determined in the same method as in Example 1. The results are shown in Table 11.

[Comparative Example 2: evaluation of PET spun yarn] For comparison, a single spun yarn (PET spun yarn) composed of commercially available 100% PET and having a fineness of 30 Nm was purchased. The water shrinkage test was performed in the same method as in Example 1, and the shrinkage rate of the PET spun yarn was determined. The results are shown in Table 11.

[Table 11]

	Fineness (Nm)	Single yarn/twin yarn	Before shrinking (cm)	After shrinking (cm)	Shrinkage rate	Note
Example 1	30	Single yarn	60	50	16.7%	Crimped staple/uncrimped staple=7/3
Example 2	48	Twin yarn	60	40.2	33.0%	Uncrimped staple	100%
Comparative Example 1	3	Single yarn	60	59.4	1%	Crimped staple	100%
Comparative Example 2	30	Single yarn	60	59.5	0.8%	-

The artificial fibroin spun yarn of the present invention had a sufficiently high shrinkage rate and the high-shrinkage artificial fibroin spun yarn after shrinkage had excellent tactile feel and flexibility. In addition, since the high-shrinkage artificial fibroin spun yarn of the present invention could be manufactured by bringing an artificial fibroin spun yarn into contact with water having a temperature lower than a boiling point and as necessary performing drying after contact with water, it could be manufactured safely.

[Test Example 2: manufacture of modified fibroin]

A modified fibroin (PRT918) having an amino acid sequence shown in SEQ ID NO 37, a modified fibroin (PRT966) having an amino acid sequence shown in SEQ ID NO 40, and a modified fibroin (PRT799) having an amino acid sequence shown in SEQ ID NO 15 were designed. A nucleic acid that encodes the designed modified fibroins was synthesized. In the nucleic acid, an NdeI site was added to the 5' end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the same nucleic acid was treated with restriction enzymes NdeI and EcoRI and cut, and then recombined into a protein expression vector pET-22b(+) to obtain an expression vector.
E. coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in a 2 mL LB culture medium containing ampicillin for 15 hours. The culture solution was added to a 100 mL culture medium containing ampicillin for seed culturing (Table 12) so that the OD₆₀₀ became 0.005. The temperature of the culture solution was maintained at 30°C and flask culture was performed until the OD₆₀₀ became 5 (about 15 hours), and thereby a seed culture solution was obtained. [Table 12]

Culture medium for seed culturing

Sample Concentration (g/L)

Glucose 5.0

KH₂PO₄ 4.0

K₂HPO₄ 9.3

Yeast Extract 6.0

Ampicillin 0.1
The seed culture solution was added to a jar fermenter containing a 500 mL production culture medium (Table 13) so that the OD₆₀₀ became 0.05. The temperature of the culture solution was maintained at 37°C and the culture was performed under control with a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved saturated oxygen concentration. [Table 13]

Production culture medium

Sample Concentration (g/L)

Glucose 12.0

KH₂PO₄ 9.0

MgSO₄·7H₂O 2.4

Yeast Extract 15

FeSO₄·7H₂O 0.04

MnSO₄·5H₂O 0.04

CaCl₂·2H₂O 0.04

ADEKA NOL (ADEKA, LG-295S) 0.1 (mL/L)
Immediately after glucose in the production culture 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 temperature of the culture solution was maintained at 37°C and the culture was performed under control with a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved saturated oxygen concentration, and the culture was performed for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution so that the final concentration was 1 mM, and thereby a modified fibroin was expressed and induced. When 20 hours had elapsed after IPTG was added, the culture solution was centrifuged, and bacteria were collected. SDS-PAGE was performed using bacteria prepared from the culture solution before IPTG was added and after IPTG was added, and the expression of the desired modified fibroin was confirmed according to the appearance of a band with a size corresponding to the desired modified fibroin depending on the addition of IPTG.
The bacteria collected 2 hours after IPTG was added were washed with a 20 mM Tris-HCl buffer (pH 7.4). The bacteria after washing were suspended in a 20 mM Tris-HCl buffer solution (pH 7.4) containing about 1 mM PMSF, and cells were crushed using a high pressure homogenizer (commercially available from GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer solution (pH 7.4) until it became highly pure. The precipitate after washing was suspended in a 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so that the concentration became 100 mg/mL, and stirred with a stirrer at 60°C for 30 minutes and dissolved. After the dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 commercially available from Sanko Junyaku Co., Ltd.). White aggregated proteins obtained after dialysis were collected through centrifugation, water was removed in a freeze dryer, and lyophilized powders were collected to obtain modified fibroins (PRT918, PRT966 and PRT799).
PRT918 and PRT966 were hydrophobic modified fibroins having an average HI of greater than 0. PRT799 was a hydrophilic modified fibroin having an average HI of 0 or less.

[Reference Example 2: evaluation of flame retardancy of modified fibroin]

Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass% was prepared as a solvent, and the lyophilized powder of the modified fibroin (PRT799) obtained in Test Example 2 was added thereto at a concentration of 24 mass%, and dissolved using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
The prepared spinning stock solution was filtered at 90°C with a metal filter having an opening of 5 µm, and then left in a 30 mL stainless syringe, bubbles were removed, and then the solution was discharged from a solid nozzle having a needle diameter of 0.2 mm to a 100 mass% methanol solidification tank. The discharge temperature was 90°C. After coagulation, the obtained raw yarn was wound and naturally dried to obtain a modified fibroin fiber (raw fiber).
A knitted fabric was manufactured using a twisted yarn obtained by twisting the obtained raw fibers according to circular knitting using a circular knitting machine. The knitted fabric had a thickness of 180 denier and a gauge number of 18. 20 g was cut out from the obtained knitted fabric to prepare a test piece.

The flammability test was performed according to a test method for powder granules or a synthetic resin having a low melting point, Fire Department Dangerous Goods Regulation Division Fire Department No. 50, May 31, 1995. The test was performed under conditions of a temperature of 22°C, a relative humidity of 45%, and an atmospheric pressure of 1,021 hPa. Table 14 shows the measurement results (oxygen concentration (%), combustion rate (%), and conversion combustion rate (%)).

[Table 14]

Oxygen concentration (%)	Combustion rate (%)	Conversion combustion rate (%)
20.0	39.1	40.1
27.0	48.1	49.3
28.0	51.9	53.2
30.0	53.6	54.9
50.0	61.2	62.7
70.0	91.1	93.3
100.0	97.6	100.0

As a result of the flammability test, the limit oxygen index (LOI) value of the knitted fabric knitted with the modified fibroin (PRT799) fiber was 27.2. Generally, when the LOI value was 26 or more, it was considered that the component had a flame retardancy. It was found that the modified fibroin had an excellent flame retardancy.

[Reference Example 3: Evaluation of hygroscopic heat generation characteristics of modified fibroin]

Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass% was prepared as a solvent, and the lyophilized powder of the modified fibroin obtained in Test Example 2 was added thereto at a concentration of 24 mass%, and dissolved using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
The prepared spinning stock solution was filtered at 60°C with a metal filter having an opening of 5 µm, and then left in a 30 mL stainless syringe, bubbles were removed, and then the solution was discharged from a solid nozzle having a needle diameter of 0.2 mm to a 100 mass% methanol solidification tank. The discharge temperature was 60°. After coagulation, the obtained raw yarn was wound and naturally dried to obtain a modified fibroin fiber (raw fiber).
For comparison, regarding raw fibers, commercially available wool fibers, cotton fibers, Tencel fibers, rayon fibers and polyester fibers were prepared.
Knitted fabrics were manufactured using the raw fibers according to flat knitting using a flat knitting machine. The knitted fabric obtained using the PRT918 fibers as raw fibers had a thickness of 1/30 N (wool count single yarn) and a gauge number of 18. The knitted fabric obtained using the PRT799 fibers as raw fibers had a thickness of 1/30 N (wool count single yarn) and a gauge number of 16. The thickness and the gauge number of the knitted fabric obtained using other raw fibers were adjusted such that the cover factor was almost the same as that of the knitted fabric obtained using the PRT918 fibers and the PRT799 fibers. The details were as follows.

Wool thickness: 2/30 N (twin yarn), gauge number: 14
Cotton thickness: 2/34 N (twin yarn), gauge number: 14
Tencel thickness: 2/30 N (twin yarn), gauge number: 15
Rayon thickness: 1/38 N (single yarn), gauge number: 14
Polyester thickness: 1/60 N (single yarn), gauge number: 14

Two pieces of knitted fabric cut into 10 cm×10 cm were combined, and four sides were sewn together to prepare a test piece (sample). The test piece was left in a low humidity environment (a temperature of 20±2°C and a relative humidity of 40±5%) for 4 hours or longer, and then moved to a high humidity environment (a temperature of 20±2°C and a relative humidity of 90±5%), and the temperature was measured using a temperature sensor attached to the center inside the test piece for 30 minutes at 1-minute intervals.
The maximum hygroscopic heat generation was determined from the measurement results according to the following Formula A. $Maximum hygroscopic heat generation = \{(maximum value of sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment) - (sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment)\} (° C) / sample weight (g)$
FIG. 7 is a graph showing an example of results of a hygroscopic and exothermic test. In the graph, the horizontal axis represents the time (min) for which the sample was left in the high humidity environment when the time at which the sample was moved from the low humidity environment to the high humidity environment was set as 0. In the graph, the vertical axis represents the temperature measured using the temperature sensor (sample temperature). In the graph shown in FIG. 7, the point indicated by M corresponds to the maximum value of the sample temperature.
Table 15 shows calculation results of the maximum hygroscopic heat generation. [Table 15]

Raw fiber Maximum hygroscopic heat generation (°C/g)

PRT918 0.040

PRT799 0.031

Wool 0.020

Cotton 0.021

Tencel 0.018

Rayon 0.025

Polyester 0.010
As shown in Table 15, it was found that the modified fibroin (PRT918 and PRT799) had a higher maximum hygroscopic heat generation and a superior hygroscopic heat generation characteristics than materials in the related art.

[Reference Example 4: evaluation of heat retention property of modified fibroin]

Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass% was prepared as a solvent, and the lyophilized powder of the modified fibroin obtained in Test Example 2 was added thereto at a concentration of 24 mass%, and dissolved using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
The prepared spinning stock solution was filtered at 60°C with a metal filter having an opening of 5 µm, and then left in a 30 mL stainless syringe, bubbles were removed, and then the solution was discharged from a solid nozzle having a needle diameter of 0.2 mm to a 100 mass% methanol solidification tank. The discharge temperature was 60°C. After coagulation, the obtained raw yarn was wound and naturally dried to obtain a modified fibroin fiber (raw fiber).
For comparison, regarding raw fibers, commercially available wool fibers, silk fibers, cotton fibers, rayon fibers and polyester fibers were prepared.
Knitted fabrics were manufactured using the raw fibers according to flat knitting using a flat knitting machine. The knitted fabric obtained using the PRT966 fibers as raw fibers had a yarn number of 30 Nm, a number of twists of 1, a gauge number of 18GG, and a basis weight of 90.1 g/m². The knitted fabric obtained using the PRT799 fibers as raw fibers had a yarn number of 30 Nm, a number of twists of 1, a gauge number of GG: 16 and a basis weight of 111.0 g/m². The thickness and the gauge number of the knitted fabric obtained using other raw fibers were adjusted such that the cover factor was almost the same as that of the knitted fabric obtained using the PRT966 fiber and the PRT799 fibers. The details were as follows.

Wool yarn number: 30 Nm, number of twists: 2, gauge number: 14GG, basis weight: 242.6 g/m²
Silk yarn number: 60 Nm, number of twists: 2, gauge number: 14GG, basis weight: 225.2 g/m²
Cotton yarn number: 34 Nm, number of twists: 2, gauge number: 14GG, basis weight: 194.1 g/m²
Rayon yarn number: 38 Nm, number of twists: 1, gauge number: 14GG, basis weight: 181.8 g/m²
Polyester yarn number: 60 Nm, number of twists: 1, gauge number: 14GG, basis weight: 184.7 g/m²

The heat retention property was evaluated using a KES-F7 Thermo Lab II tester (commercially available from Kato Tech Co., Ltd.) and a dry contact method (a method assuming that the skin and clothes were in direct contact with each other in a dry state). One piece of knitted fabric cut into 20 cm×20 cm was prepared as a test piece (sample). The test piece was set on a hot plate set to a certain temperature (30°C) and the amount of heat (a) dissipated through the test piece was determined under conditions of a wind velocity in a wind tunnel of 30 cm/sec. The amount of heat (b) dissipated was determined under the same conditions as above without setting the test piece, and the heat retention rate (%) was calculated according to the following formula. $Heat retention rate (%) = (1 - a / b) \times 100$
The heat retention index was determined from the measurement results according to the following Formula B. $heat retention index = heat retention rate (%) / basis weight (g / m^{2}) of sample$
Table 16 shows the calculation results of the heat retention index. A material having a higher heat retention index can be evaluated as having a superior heat retention property. [Table 16]

Raw fiber Heat retention index

PRT966 0.33

PRT799 0.22

Wool 0.16

Silk 0.11

Cotton 0.13

Rayon 0.02

Polyester 0.18
As shown in Table 16, it was found that the modified fibroins (PRT966 and PRT799) had a higher heat retention index and a superior heat retention property than materials in the related art.

Reference Signs List

1 Extrusion device
2 Unstretched yarn manufacturing device
3 Wet heat stretching device
4 Drying device
6 Dope solution
10 Spinning device
20 Coagulating liquid tank
21 Stretched bathtub
36 Modified fibroin fiber (filament)

Claims

A high-shrinkage artificial fibroin spun yarn which is a shrunk artificial fibroin spun yarn including a modified fibroin and having a shrinkage rate of greater than 1% defined by the following Formula I: $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$
The high-shrinkage artificial fibroin spun yarn according to claim 1,
wherein the modified fibroin is a modified spider silk fibroin.
The high-shrinkage artificial fibroin spun yarn according to claim 1 or 2, which is shrunk when brought into contact with water having a temperature lower than a boiling point.
The high-shrinkage artificial fibroin spun yarn according to claim 3,
wherein the temperature of the water is 10 to 90°C.
The high-shrinkage artificial fibroin spun yarn according to claim 3 or 4, which is additionally shrunk when drying is performed after contact with the water.
A method for manufacturing a high-shrinkage artificial fibroin spun yarn, comprising:
a shrinkage process in which an artificial fibroin spun yarn including a modified fibroin is brought into contact with water having a temperature lower than a boiling point,

wherein a shrinkage rate defined by the following Formula I is greater than 1%: $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$
The method for manufacturing a high-shrinkage artificial fibroin spun yarn according to claim 6,
wherein the modified fibroin is a modified spider silk fibroin.
The method for manufacturing a high-shrinkage artificial fibroin spun yarn according to claim 6 or 7,
wherein the temperature of the water is 10 to 90°C.
The method for manufacturing a high-shrinkage artificial fibroin spun yarn according to any one of claims 6 to 8,
wherein the shrinkage process further includes drying of the artificial fibroin spun yarn after it is brought into contact with the water.
A method for shrinking an artificial fibroin spun yarn, comprising:
a shrinkage process in which an artificial fibroin spun yarn including a modified fibroin is brought into contact with water having a temperature lower than a boiling point,

wherein a shrinkage rate defined by the following Formula I is greater than 1%: $Shrinkage rate = \{1 - (length of shrunk artificial fibroin spun yarn / length of artificial fibroin spun yarn after spinning and before contact with water)\} \times 100 (%)$
The method for shrinking an artificial fibroin spun yarn according to claim 10,
wherein the modified fibroin is a modified spider silk fibroin.
The method for shrinking an artificial fibroin spun yarn according to claim 10 or 11,
wherein the temperature of the water is 10 to 90°C.
The method for shrinking an artificial fibroin spun yarn according to any one of claims 10 to 12,
wherein the shrinkage process further includes drying of the artificial fibroin spun yarn after it is brought into contact with the water.
An artificial fibroin spun yarn including a modified fibroin and having a shrinkage rate of greater than 1% defined by the following Formula II: $Shrinkage rate = \{1 - (length of artificial fibroin spun yarn subjected to shrinkage processing including being brought into contact with water having a temperature lower than a boiling point / length of artificial fibroin spun yarn before shrinkage processing is performed)\} \times 100 (%)$
The high-shrinkage artificial fibroin spun yarn according to any one of claims 1 to 5,
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein the domain sequence includes an amino acid sequence with a reduced content of the (A)_n motif, which corresponds to deletion of at least one or more (A)_n motifs, as compared with a naturally derived fibroin:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
The high-shrinkage artificial fibroin spun yarn according to any one of claims 1 to 5,
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein the domain sequence includes an amino acid sequence with a reduced content of glycine residues, which corresponds to substitution of at least one or more glycine residues in REP with other amino acid residues, as compared with a naturally derived fibroin:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
The high-shrinkage artificial fibroin spun yarn according to any one of claims 1 to 5,
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m, and
wherein the domain sequence includes an amino acid sequence including a region having a locally large hydrophobicity index, which corresponds to substitution of one or more amino acid residues in REP with an amino acid residue having a large hydrophobicity index and/or insertion of one or more amino acid residues having a large hydrophobicity index into REP, as compared with a naturally derived fibroin:
[in Formula 1, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
The high-shrinkage artificial fibroin spun yarn according to any one of claims 1 to 5,
wherein the modified fibroin includes a domain sequence represented by Formula 1: [(A)_n motif-REP]_m or Formula 2: [(A)_n motif-REP]_m-(A)_n motif, and
wherein the domain sequence includes an amino acid sequence with a reduced content of glutamine residues, which corresponds to deletion of one or more glutamine residues in REP or substitution with other amino acid residues, as compared with a naturally derived fibroin:
[in Formula 1 and in Formula 2, the (A)_n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A)_n motif is 80% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer of 2 to 300; a plurality of (A)_n motifs may have the same amino acid sequence or different amino acid sequences; and a plurality of REPs may have the same amino acid sequence or different amino acid sequences].
The high-shrinkage artificial fibroin spun yarn according to any one of claims 1 to 5,
wherein the modified fibroin has a limit oxygen index (LOI) value of 26.0 or more.
The high-shrinkage artificial fibroin spun yarn according to any one of claims 1 to 5,
wherein the modified fibroin has a maximum hygroscopic heat generation of greater than 0.025°C/g determined according to the following Formula A: $Maximum hygroscopic heat generation = \{(maximum value of sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment) - (sample temperature when sample is left in low humidity environment until sample temperature reaches equilibrium and is then moved to high humidity environment)\} (° C) / sample weight (g)$
[in Formula A, a low humidity environment means an environment of a temperature of 20°C and a relative humidity of 40%, and a high humidity environment means an environment of a temperature of 20°C and a relative humidity of 90%].