JP7340262B2 - High-shrinkage artificial fibroin spun yarn and its manufacturing method, and artificial fibroin spun yarn and its shrinkage method - Google Patents

Description

The present invention relates to a highly shrinkable artificial fibroin spun yarn and a method for producing the same, as well as an artificial fibroin spun yarn and a method for shrinking the same.

Conventionally, spun yarns made of synthetic fibers, natural fibers, etc. have been used as materials for various knitted fabrics. When producing knitted fabrics, spun yarns that can meet the requirements for knitted fabrics are selected. For example, when manufacturing clothing, bedding, and the like that are highly soft to the touch and have a luxurious feel, spun silk yarn or the like is selected as the material.

Further, depending on the characteristics required of the knitted fabric, the spun yarn used as the material may be subjected to predetermined processing. For example, in clothing, bedding, etc., when higher flexibility and heat retention are desired in addition to high texture and luxury, spun silk yarn that has been subjected to shrink processing to increase bulkiness is used. It may happen.

As a method for shrinking silk, for example, the so-called salt shrinking process is known, in which silk is immersed in an aqueous solution (salt shrink solution) in which inorganic salts such as calcium nitrate and calcium chloride are dissolved at high concentrations, and then shrunk ( For example, Patent Document 1).

On the other hand, for example, synthetic fibers such as polyester fibers, polyamide fibers, and acrylic fibers commonly used in clothing, bedding, etc. achieve a shrinkage rate of 40% or more when brought into contact with boiling water (Patent Document 2).

Japanese Patent Application Publication No. 2001-64866 Japanese Patent Application Publication No. 2009-121003

However, the shrinkage rate of silk was extremely small when it came into contact with only water. Further, the shrinkage method described in Patent Document 2 involves great danger because it handles high-temperature boiling water.

An object of the present invention is to provide a high-shrinkage artificial fibroin spun yarn that has a sufficiently high shrinkage rate, is excellent in texture and flexibility, and can be manufactured safely, and a method for manufacturing the same. Another object of the present invention is to provide an artificial fibroin spun yarn that can safely produce a highly shrinkable artificial fibroin spun yarn with a sufficiently high shrinkage rate, and a method for shrinking the same.

The present invention relates to the following inventions, for example.
[1]
A shrunken artificial fibroin yarn comprising modified fibroin, the yarn comprising:
A high shrinkage artificial fibroin spun yarn having a shrinkage rate defined by the following formula I of 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)} x 100 (%) ... (Formula I)
[2]
The highly contractile artificial fibroin spun yarn according to [1], wherein the modified fibroin is modified spider silk fibroin.
[3]
The high-shrinkage artificial fibroin spun yarn according to [1] or [2], which is shrunk by contact with water below the 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 further shrunk by drying after contact with the water.
[6]
A step of bringing an artificial fibroin spun yarn containing modified fibroin into contact with water below the boiling point to cause it to shrink,
A method for producing a highly shrinkable artificial fibroin spun yarn, which has a shrinkage rate defined by the following formula I of 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)} x 100 (%) ... (Formula I)
[7]
The method for producing a high-shrinkage artificial fibroin spun yarn according to [6], wherein the modified fibroin is modified spider silk fibroin.
[8]
The method for producing 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 producing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [8], wherein the shrinking step further includes drying the artificial fibroin spun yarn after contact with the water.
[10]
A step of bringing an artificial fibroin spun yarn containing modified fibroin into contact with water below the boiling point to cause it to shrink,
A method for shrinking an artificial fibroin spun yarn, wherein 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)} x 100 (%) ... (Formula I)
[11]
The method for shrinking artificial fibroin spun yarn according to [10], wherein the modified fibroin is modified spider silk fibroin.
[12]
The method for shrinking 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 shrinking step further includes drying the artificial fibroin spun yarn after contact with the water.
[14]
An artificial fibroin spun yarn containing modified fibroin and having a shrinkage rate defined by the following formula II of more than 1%.
Shrinkage rate = {1 - (Length of artificial fibroin spun yarn subjected to shrinkage processing including contact with water below the boiling point / Length of artificial fibroin spun yarn before applying the above shrinkage treatment)} x 100 (% )...(Formula II)
[15]
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
The domain sequence has an amino acid sequence with a reduced content of (A) _n motifs, which corresponds to the deletion of at least one or more (A) _n motifs compared to naturally-derived fibroin. High shrinkage artificial fibroin spun yarn according to any one of [1] to [5], method for producing high shrinkage artificial fibroin spun yarn according to any one of [6] to [9], [10] to [13] The method for shrinking an artificial fibroin spun yarn according to any one of [14] or the artificial fibroin spun yarn according to [14].
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 83% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ]
[16]
The above domain sequence corresponds to the deletion of at least one (A) _n motif for every one to three (A) _n motifs from the N-terminus to the C-terminus compared to naturally-derived fibroin. The high-shrinkage artificial fibroin spun yarn, the method for producing 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], which has an amino acid sequence of [15].
[17]
The above domain sequence has at least two consecutive (A) _n- motif deletions from the N-terminal side to the C-terminal side, and one (A) _n- motif deletion compared to naturally-derived fibroin. The high-shrinkage artificial fibroin spun yarn, the method for producing 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], which has an amino acid sequence corresponding to repeated sequences in this order. .
[18]
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
From the N-terminal side to the C-terminal side, compare the number of amino acid residues in the REPs of two adjacent [(A) _n motif-REP] units sequentially to determine the number of amino acid residues in the REP with fewer amino acid residues. is 1, the sum of the number of amino acid residues in two adjacent [(A) _n motif-REP] units where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3. The highly contractile artificial construct according to any one of [1] to [5], wherein x/y is 50% or more, where x is the maximum value and y is the total number of amino acid residues in the domain sequence. Fibroin spun yarn, the method for producing a highly shrinkable 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 [ [14] The artificial fibroin spun yarn described in [14].
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 83% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ]
[19]
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
The domain sequence has a reduced content of glycine residues, which corresponds to substitution of at least one or more glycine residues in REP with another amino acid residue, as compared to naturally occurring fibroin. The high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], having an amino acid sequence, the method for producing the high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], [10] ] to [13], or the artificial fibroin spun yarn according to [14].
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 83% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ]
[20]
Compared to naturally occurring fibroin, the domain sequence has at least one or more motif sequences selected from GGX and GPGXX (where X represents an amino acid residue other than glycine) in REP. Production of the high-shrinkage artificial fibroin spun yarn, the high-shrinkage artificial fibroin spun yarn according to [19], which has an amino acid sequence corresponding to one glycine residue in the motif sequence being substituted with another amino acid residue method, method for shrinking artificial fibroin yarn, or artificial fibroin yarn.
[21]
The high-shrinkage artificial fibroin spun yarn and high-shrinkage artificial fibroin spun yarn according to [20], wherein the proportion of motif sequences in which glycine residues are substituted with other amino acid residues is 10% or more of the total motif sequences. A method for producing yarn, a method for shrinking artificial fibroin spun yarn, or an artificial fibroin spun yarn.
[22]
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
XGX contained in all REPs in the sequence excluding the sequence from the (A) _n motif located on the C-terminal side of the above domain sequence to the C-terminus of the above domain sequence (where X is an amino acid residue other than glycine) The total number of amino acid _residues of the amino acid sequence consisting of The highly contractile artificial fibroin spun yarn according to any one of [1] to [5], wherein z/w is 50.9% or more, where w is the total number of amino acid residues, [6] to [ The method for producing a highly shrinkable artificial fibroin spun yarn according to any one of [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] .
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 83% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ]
[23]
The above-mentioned modified fibroin corresponds to one or more glycine residues in REP being substituted with another amino acid residue, as compared to naturally-derived fibroin, and one or more additional amino acid residues. The high-shrinkage artificial fibroin spun yarn according to any one of [19] to [22], having an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of , and the method for producing a high-shrinkage artificial fibroin spun yarn , a method for shrinking an artificial fibroin yarn, or an artificial fibroin yarn.
[24]
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
In the above domain sequence, one or more amino acid residues in REP are substituted with amino acid residues having a larger hydrophobicity index than that of naturally occurring fibroin, and/or one or more hydrophobic residues in REP The highly contractile artificial fibroin fabric according to any one of [1] to [5], which has an amino acid sequence that includes a region with a locally large hydrophobicity index, which corresponds to the insertion of an amino acid residue with a large sex index. Yarn, the method for producing 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 [14] The artificial fibroin spun yarn described in .
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 83% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ]
[25]
The high-shrinkage artificial fibroin spun yarn, the method for producing a high-shrinkage artificial fibroin spun yarn, the artificial Method for shrinking fibroin spun yarn or artificial fibroin spun yarn.
[26]
[ 24] or [25], the high-shrinkage artificial fibroin spun yarn, the method for producing a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial fibroin spun yarn, or the artificial fibroin spun yarn.
[27]
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
Average hydrophobicity index of four consecutive amino acid residues in all REPs included in the sequence from the domain sequence ( _A ) located at the most C-terminal side to the C-terminus of the domain sequence above. The total number of amino acid residues included in the region with a value of 2.6 or more is p, and the sequence from the (A) _n motif located closest to the C-terminus to the C-terminus of the above domain sequence was removed from the above domain sequence. The highly contractile artificial fibroin spun yarn according to any one of [1] to [5], wherein p/q is 6.2% or more, where q is the total number of amino acid residues included in the sequence, [6 ] to [9], the method for producing a highly shrinkable artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin yarn according to [14]. Fibroin spun yarn.
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 83% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ]
[28]
The above-mentioned modified fibroin has one or more amino acid residues in REP replaced with amino acid residues having a larger hydrophobicity index than naturally-derived fibroin, and/or one or more hydrophobic residues in REP. In addition to the insertion of an amino acid residue with a large sex index, the amino acid sequence corresponds to the substitution, deletion, insertion, and/or addition of one or more amino acid residues [24 ] - [27] High shrinkage artificial fibroin spun yarn, method for producing high shrinkage artificial fibroin spun yarn, method for shrinking artificial fibroin spun yarn, or artificial fibroin spun yarn according to any one of [27].
[29]
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif,
The content of glutamine residues in the above domain sequence corresponds to the deletion of one or more glutamine residues in REP or substitution with other amino acid residues compared to naturally occurring fibroin. Production of the high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], which has an amino acid sequence with reduced method, 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].
[In Formula 1 and Formula 2, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 80% of the total number of amino acid residues in (A) _n motif. That's all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ]
[30]
The above-mentioned modified fibroin contains a GPGXX (wherein, X indicates an amino acid residue other than a glycine residue) motif in REP, and has a GPGXX motif content of 10% or more, the highly contractile artificial material according to [29]. A fibroin spun yarn, a method for producing a highly shrinkable artificial fibroin spun yarn, a method for shrinking an artificial fibroin spun yarn, or an artificial fibroin spun yarn.
[31]
The modified fibroin has a glutamine residue content of 9% or less, the high-shrinkage artificial fibroin spun yarn, the method for producing a high-shrinkage artificial fibroin spun yarn, and the shrinkage of the artificial fibroin spun yarn according to [29] or [30]. method, or artificial fibroin spun yarn.
[32]
The other amino acid residues mentioned above are isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine (G), threonine ( Any of [29] to [31], which is an amino acid residue selected from the group consisting of T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). The high-shrinkage artificial fibroin spun yarn, the method for producing a high-shrinkage artificial fibroin spun yarn, the method for shrinking the artificial fibroin spun yarn, or the artificial fibroin spun yarn described in .
[33]
The modified fibroin has a REP hydrophobicity of -0.8 or more, the high-shrinkage artificial fibroin spun yarn according to any one of [29] to [32], the method for producing a high-shrinkage artificial fibroin spun yarn, A method for shrinking artificial fibroin spun yarn, or an artificial fibroin spun yarn.
[34]
The above-mentioned modified fibroin has one or more glutamine residues deleted or substituted with other amino acid residues in REP compared to naturally-derived fibroin, and also has one or more glutamine residues substituted with other amino acid residues. The high-shrinkage artificial fibroin spun yarn or high-shrinkage artificial fibroin according to any one of [29] to [33], which has an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of multiple amino acid residues. A method for producing a spun yarn, a method for shrinking an artificial fibroin yarn, or an artificial fibroin yarn.
[35]
The modified fibroin has a limiting oxygen index (LOI) value of 26.0 or more, the high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], or any one of [6] to [9]. The method for producing a highly shrinkable artificial fibroin spun yarn according to [10] to [13], or the artificial fibroin spun yarn according to [14].
[36]
The modified fibroin has a maximum hygroscopic exothermic value determined according to the following formula A of more than 0.025°C/g, the high shrinkage artificial fibroin spun yarn according to any one of [1] to [5], [6] to The method for producing a highly shrinkable artificial fibroin spun yarn according to any one of [9], the method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spinning according to [14] thread.
Formula A: Maximum hygroscopic exotherm = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then transferred to a high humidity environment) - (The sample temperature is transferred to a high humidity environment) Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (°C) / Sample weight (g)
[In Formula A, the low humidity environment means an environment with a temperature of 20° C. and a relative humidity of 40%, and the high humidity environment means an environment with a temperature of 20° C. and a relative humidity of 90%. ]

According to the present invention, it is possible to provide a high-shrinkage artificial fibroin spun yarn that has a sufficiently high shrinkage rate, is excellent in texture and flexibility, and 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 that can safely produce a highly shrinkable artificial fibroin spun yarn with a sufficiently high shrinkage rate, and a method for shrinking the same.

FIG. 2 is a schematic diagram showing the domain sequence of modified fibroin according to one embodiment. FIG. 2 is a diagram showing the distribution of z/w (%) values of naturally-derived fibroin. FIG. 2 is a diagram showing the distribution of x/y (%) values of naturally-derived fibroin. FIG. 2 is a schematic diagram showing the domain sequence of modified fibroin according to one embodiment. FIG. 2 is a schematic diagram showing the domain sequence of modified fibroin according to one embodiment. FIG. 1 is an explanatory diagram schematically showing an example of a spinning device for producing modified fibroin fibers (filaments). It is a graph which shows an example of the result of a moisture absorption heat generation test.

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated in detail. However, the present invention is not limited to the following embodiments.

[High shrinkage artificial fibroin spun yarn]
The highly contractible artificial fibroin spun yarn according to the present embodiment is a contracted artificial fibroin fiber containing modified fibroin. The high shrinkage artificial fibroin spun yarn according to the present embodiment has a shrinkage rate defined by the following formula I of 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)} x 100 (%) ... (Formula I)

<Modified fibroin>
The modified fibroin according to the present embodiment has a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m - (A) _n motif. It is a protein that contains The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and C-terminal side of the domain sequence. Although the N-terminal sequence and the C-terminal sequence are not limited thereto, typically they are regions that do not have repeated amino acid motifs characteristic of fibroin, and consist of about 100 amino acid residues.

As used herein, "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be a fibroin whose domain sequence is the same as the amino acid sequence of naturally occurring fibroin. "Naturally derived fibroin" as used herein is also represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif. It is a protein that contains a domain sequence that is

"Modified fibroin" may be one that uses the amino acid sequence of naturally-derived fibroin as is, or one that relies on the amino acid sequence of naturally-derived fibroin and has its amino acid sequence modified (for example, a cloned naturally-derived fibroin). The amino acid sequence may be modified by modifying the gene sequence of fibroin), or it may be artificially designed and synthesized without relying on naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence). The desired amino acid sequence may be obtained by chemical synthesis). In addition, as the modified fibroin according to this embodiment, modified spider silk fibroin is preferably used because it is excellent in heat retention, moisture absorption and heat generation properties, and/or flame retardancy.

In this specification, the term "domain sequence" refers to the crystalline region (typically, corresponding to the (A) _n motif of the amino acid sequence) unique to fibroin and the amorphous region (typically, corresponding to the REP of the amino acid sequence). ), and is an amino acid sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif. means an array. Here, the (A) _n motif indicates an amino acid sequence consisting mainly of alanine residues, and the number of amino acid residues is 2 to 27. (A) The number of amino acid residues in _{the n} motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. . In addition, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) _n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A) _n motifs present in a plurality in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP is an amino acid sequence composed of 10-200, 10-180, 10-160, 10-140, 10-120, 10-100, 10-80, 10-60, or 10-40 amino acid residues. You can. m represents an integer of 2-300, 8-300, 10-300, 20-300, 40-300, 60-300, 80-300, 10-200, 20-200, 20-180, 20-160, It may be an integer from 20 to 140 or from 20 to 120. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences.

The modified fibroin according to the present embodiment has an amino acid sequence corresponding to, for example, substitution, deletion, insertion, and/or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. It can be obtained by modifying . Substitutions, deletions, insertions and/or additions of amino acid residues can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.

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

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

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

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

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

A more specific example of naturally-derived fibroin includes fibroin whose sequence information is registered in NCBI GenBank. For example, among sequence information registered in NCBI GenBank that includes INV as DIVISION, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are listed as keywords in DEFINITION. This can be confirmed by extracting the sequence, the character string of a specific product from the CDS, and the sequence in which the specific character string is written in TISSUE TYPE from the SOURCE.

The modified fibroin according to the present embodiment may be modified silk fibroin (the amino acid sequence of silk protein produced by silkworms is modified), or modified spider silk fibroin (the amino acid sequence of silk protein produced by spiders is modified). (modified amino acid sequence) may also be used. As the modified fibroin, modified spider silk fibroin is preferable because it has excellent heat retention properties, moisture absorption heat generation properties, and/or flame retardance.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large retractor dragline protein produced in the large flial gland of spiders, and a domain sequence with a reduced content of glycine residues. a modified fibroin having a domain sequence with a reduced content of (A) _n motif (a third modified fibroin), a content of glycine residues, and (A) _n Modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin with a domain sequence including a region with a locally large hydrophobicity index (fifth modified fibroin), and content of glutamine residues modified fibroin having a reduced domain sequence (sixth modified fibroin).

The first modified fibroin includes a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, the number of amino acid residues in the (A) _n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, and an integer of 10 to 20. is even more preferred, an integer of 4 to 16 is even more preferred, an integer of 8 to 16 is particularly preferred, and an integer of 10 to 16 is most preferred. In the first modified fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. It is more preferable that the number of residues is 20 to 75, and even more preferable that the number of residues is 20 to 75. The first modified fibroin has amino acid residues in which the total number of glycine residues, serine residues, and alanine residues contained in the amino acid sequence represented by formula 1: [(A) _n motif-REP] _m It is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more of the total number.

The first modified fibroin contains an amino acid sequence unit represented by formula 1: [(A) _n motif-REP] _m , and has an amino acid sequence whose C-terminal sequence is shown in any of SEQ ID NOs: 1 to 3, or It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of the C-terminal 50 amino acid residues of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is the same as that of ADF3 (GI: 1263287, NCBI). The amino acid sequence shown in SEQ ID NO: 3 is the same as that obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 1 is obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1. Identical to the amino acid sequence.

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

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

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

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

The second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine), compared to naturally-derived fibroin. ) has an amino acid sequence corresponding to the substitution of one glycine residue in at least one or more of the motif sequences with another amino acid residue. It's okay.

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

The second modified fibroin includes a domain sequence represented by formula 1: [(A) _n motif-REP] _m , and from the above domain sequence, from the (A) _n motif located at the most C-terminal side to the above domain sequence. Let z be the total number of amino acid residues in the amino acid sequence consisting of z/w is 30% or more, where w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the above domain sequence, It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues), even more preferably.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The modified fibroin (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin (2-iv) is also a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

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

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

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

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

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

The third modified fibroin has at least two consecutive domain sequences from the N-terminus to the C-terminus (A), a deletion of _one (A) ) It may have an amino acid sequence corresponding to deletions of _{the n} motif repeated in this order.

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

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

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

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

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

In Figure 1, when the one with the smaller number of amino acid residues is set as 1, the set of [(A) _n motif-REP] units where the ratio of the number of amino acid residues on the other side is 1.8 to 11.3 is Indicated by a solid line. In this specification, this ratio is referred to as the jagged ratio. When the one with the smaller number of amino acid residues is set as 1, the pair of [(A) _n motif-REP] units in which the ratio of the number of amino acid residues of the other one is less than 1.8 or more than 11.3 is indicated by a broken line. Indicated.

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

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

In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, and even more preferably 70% or more. It is preferably at least 75%, even more preferably at least 80%, particularly preferably at least 80%. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x/y is preferably 89.6% or more. /y is preferably 77.1% or more, and when the jagged ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the jagged ratio is 1:1.9 to 8.4. : 1.9 to 4.1, x/y is preferably 64.2% or more.

If the third modified fibroin is a modified fibroin in which there is a plurality of modified fibroin in the domain sequence (A) in which at least seven of _{the n} motifs are composed of only alanine residues, x/y shall be 46.4% or more. is preferable, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferable that there be. There is no particular restriction on the upper limit of x/y, as long as it is 100% or less.

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

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

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

As a more specific example of the third modified fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.

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

The x/y value of the amino acid sequence shown by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) at a jagged ratio of 1:1.8 to 11.3 is 15.0%. The value of x/y in both the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 is 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%. The values of z/w in the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, and 66.9%, respectively. 1% and 70.0%.

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

The modified fibroin (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin (3-ii) is also a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

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

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

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

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

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

The modified fibroin (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin (3-iv) is also a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

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

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

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

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

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

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

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

The fifth modified fibroin is characterized in that one or more amino acid residues in REP are substituted with amino acid residues having a larger hydrophobicity index than in naturally occurring fibroin, and/or one or more amino acid residues in REP In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, one or more amino acid residues are substituted, deleted, inserted, and/or added compared to naturally-derived fibroin. There may also be modifications in the amino acid sequence corresponding to the changes made.

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

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

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

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

For example, if 20 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" are extracted (no overlap), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2. The region with a value of .6 or more includes 20 consecutive 4 amino acid residues (no overlap), and p is 20×4=80. Also, for example, if two "four consecutive amino acid residues with an average value of hydrophobicity index of 2.6 or more" overlap by one amino acid residue, the hydrophobicity index of the four consecutive amino acid residues The region where the average value of is 2.6 or more will contain 7 amino acid residues (p=2×4−1=7. “−1” is the deduction of overlap.). For example, in the case of the domain sequence shown in Figure 4, there are 7 non-overlapping "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", so p is 7 x 4 = It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 4, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A) _n motif present at the end of the C-terminal side). Next, p/q (%) can be calculated by dividing p by q. In the case of FIG. 4, it is 28/170=16.47%.

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

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

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

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

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

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

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

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

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

More specific examples of modified fibroin containing a tag sequence include (5-iii) the amino acid sequence shown by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666); or (5-iv) ) Modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24 can be mentioned.

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

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

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

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

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

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

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

When the sixth modified fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.

In this specification, "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - ( _A ) fibroin (modified fibroin or naturally In fibroin, the number of GPGXX motifs contained in that region of all REPs contained in the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence. The total number multiplied by 3 (i.e., equivalent to the total number of G and P in the GPGXX motif) is defined as s, and the sequence from the (A) _n motif located on the C-terminal side to the C-terminus of the domain sequence is calculated from the domain sequence. The GPGXX motif content is calculated as s/t, where t is the total number of amino acid residues in all REPs excluding (A) _n motif.

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

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

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

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

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

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

As shown in Table 1, amino acid residues with a higher hydrophobic index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H) may be mentioned. can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

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

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

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

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

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

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

The amino acid sequence shown by SEQ ID NO: 30 is obtained by replacing all QQ in the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT468) with VL. The amino acid sequence shown by SEQ ID NO: 31 is obtained by replacing all QQ in the amino acid sequence shown by SEQ ID NO: 8 with VL, and replacing the remaining Q with I.

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

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

The modified fibroin (6-i) consists of the amino acid sequence shown by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. There may be.

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

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

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

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

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

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

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

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

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

The modified fibroin has at least two or more of the characteristics possessed by the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. It may also be a modified fibroin that has the following characteristics.

The modified fibroin according to the present embodiment may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, may be 28 or more, may be 29 or more, or may be 30 or more. In the present specification, the LOI value is a value measured in accordance with the test method for synthetic resins in the form of powder or granules or with a low melting point published by the Fire and Disaster Management Agency, Hazardous Materials Regulation Division, Fire Safety No. 50, May 31, 1995.

The modified fibroin according to the present embodiment may have a maximum hygroscopic exotherm determined according to the following formula A of more than 0.025° C./g.
Formula A: Maximum hygroscopic exotherm = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then transferred to a high humidity environment) - (The sample temperature is transferred to a high humidity environment) Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (°C) / Sample weight (g)
In Formula A, the low humidity environment means an environment with a temperature of 20° C. and a relative humidity of 40%, and the high humidity environment means an environment with a temperature of 20° C. and a relative humidity of 90%.

The modified fibroin according to the present embodiment may have a maximum hygroscopic exotherm of 0.026°C/g or more, 0.027°C/g or more, or 0.028°C/g or more. The temperature may be 0.029°C/g or higher, 0.030°C/g or higher, 0.035°C/g or higher, 0.040°C/g or higher. There may be. There is no particular restriction on the upper limit of the maximum hygroscopic exotherm, but it is usually 0.060° C./g or less.

The modified fibroin according to the present embodiment may have a heat retention index of 0.20 or more determined according to formula B below.
Formula B: Heat retention index = heat retention rate (%) / basis weight of sample (g/m ² )
Here, in this specification, the heat retention rate means the heat retention rate measured by the dry contact method using a Thermolab II type tester (under wind of 30 cm/sec), and by the method described in the Examples section below. It is the value to be measured.

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. It may be .30 or more, and it may be .32 or more. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.

<Method for producing modified fibroin>
The modified fibroin according to this embodiment can be obtained by, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing the nucleic acid.

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

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

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector. As the expression vector, one that is capable of autonomous replication in the host cell or can be integrated into the chromosome of the host, and contains a promoter at a position where the nucleic acid encoding the modified fibroin can be transcribed is preferably used.

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

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

When a prokaryote is used as a host, vectors for introducing a nucleic acid encoding modified fibroin include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, Examples include pNCO2 (Japanese Patent Application Laid-open No. 2002-238569).

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

When a eukaryote is used as a host, examples of vectors for introducing a nucleic acid encoding modified fibroin include YEP13 (ATCC 37115) and YEp24 (ATCC 37051). Any method for introducing DNA into the host cells can be used as a method for introducing the expression vector into the host cells. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method, and the like.

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

Modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting it from the culture medium. The host can be cultured in a culture medium according to a method commonly used for culturing hosts.

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

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

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

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

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

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

<Production method of modified fibroin fiber (filament)>
The modified fibroin fiber (filament) according to the present embodiment is obtained by spinning the modified fibroin described above, and contains the modified fibroin described above as a main component. Modified fibroin fibers can be produced by known spinning methods. That is, for example, when producing modified fibroin fibers containing modified fibroin as a main component, first, the modified fibroin produced according to the method described above is treated with dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), If necessary, it is added to a solvent such as formic acid or hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution promoter, and dissolved to prepare a dope solution. Next, using this dope solution, modified fibroin fibers can be obtained by spinning by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning. Preferred spinning methods include wet spinning or dry-wet spinning.

FIG. 6 is an explanatory diagram schematically showing an example of a spinning device for producing modified fibroin fibers. 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 undrawn 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 explained. First, the dope liquid 6 stored in the storage tank 7 is pushed out from the mouthpiece 9 by the gear pump 8. On a lab scale, the dope may be filled into a cylinder and forced out of a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied into the coagulation liquid 11 of the coagulation liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulum is formed. Next, the fibrous coagulated material is supplied into the hot water 12 in the drawing bath 21 and drawn. The stretching ratio is determined by the speed ratio of the supply nip roller 13 and the take-off nip roller 14. Thereafter, the drawn fibrous coagulate is supplied to the drying device 4 and dried in the thread path 22, and modified fibroin fibers 36 are obtained as the thread body 5. 18a to 18g are thread guides.

The coagulating liquid 11 may be any solution that can be used to remove the solvent, 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 contain water as appropriate. The temperature of the coagulating liquid 11 is preferably 0 to 30°C. When using a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm as the mouthpiece 9, the extrusion rate is preferably 0.2 to 6.0 ml/hour, and 1.4 to 4.0 ml/hour per hole. More preferably, it is time. The distance that the coagulated modified fibroin passes through the coagulation liquid 11 (substantially the distance from the thread guide 18a to the thread guide 18b) is sufficient as long as it can efficiently remove the solvent. It is 500mm. The take-off speed of the undrawn yarn may be, for example, 1 to 20 m/min, preferably 1 to 3 m/min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulation liquid 11. The coagulating liquid tank 20 may be provided in multiple stages, and stretching may be performed at each stage or at a specific stage, as necessary.

Note that the stretching performed when obtaining the modified fibroin fibers includes, for example, the pre-stretching performed in the coagulation bath 20 and the moist heat stretching performed in the stretching bath 21, as well as dry heat stretching.

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

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

The wet heat stretching and the dry heat stretching may be performed individually, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching. For example, wet heat stretching and dry heat stretching can be carried out in an appropriate combination.

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

<Production method of artificial fibroin spun yarn>
The artificial fibroin spun yarn is obtained by a method including a cutting step of cutting the above-mentioned modified fibroin fibers (filaments) into appropriate lengths to obtain modified fibroin staples, and a spinning step of spinning the obtained modified fibroin staples. be able to.

The cutting step can be performed using any device capable of cutting modified fibroin fibers. An example of such a device is a tabletop fiber cutting machine (s/NO.IT-160201-NP-300).

The length of the modified fibroin staple is not particularly limited, but is, for example, 20 mm or more, may be 20 to 140 mm, may be 70 to 140 mm, or may be 20 to 70 mm.

The spinning process can be carried out by a known spinning method. Examples of the spinning method include cotton spinning, worsting, and wool spinning. The devices used in these spinning methods are not particularly limited, and commonly used devices can be used. In addition, in the spinning process, the modified fibroin staple may first be opened or defibrated using an opener, a breaker, or the like.

The method for producing an artificial fibroin spun yarn may include a crimping step before or after the cutting step. The crimping step may be, for example, a step of implementing a mechanical crimping method such as a pressing method, in which modified fibroin fibers (filaments) or modified fibroin staples are brought into contact with an aqueous medium and crimped (hereinafter referred to as (sometimes referred to as "water crimping") process.

The aqueous medium is a liquid or gaseous (steam) medium containing water (including water vapor). The aqueous medium may be water or a mixture of water and a hydrophilic solvent. Further, as the hydrophilic solvent, it is also possible to use, for example, volatile solvents such as ethanol and methanol, or their vapors. The aqueous medium may be a liquid mixture of water and a volatile solvent such as ethanol or methanol, and is preferably a liquid mixture of water or water and ethanol. By using an aqueous medium containing a volatile solvent or its vapor, it is possible to improve the drying speed after water crimping, and it is also possible to impart a soft texture to the final crimped staple. There is. The ratio of water to the volatile solvent or its vapor is not particularly limited, and for example, the mass ratio of water to volatile solvent or its vapor may be 10:90 to 90:10. The proportion of water is preferably 30% by mass or more, and may be 40% by mass or 50% by mass or more.

The aqueous medium is preferably a liquid or gas containing water (including water vapor) at a temperature of 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, more preferably 100 to 120°C. When the steam temperature of the aqueous medium is 230° C. or lower, thermal denaturation of the modified fibroin filament can be prevented. 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 from the viewpoint of efficiently imparting crimp, and from the viewpoint of maintaining high fiber strength of the modified fibroin filament. , 60°C or less is preferable.

The time of contact with the aqueous medium is not particularly limited, but may be 30 seconds or more, 1 minute or more, or 2 minutes or more, and is preferably 10 minutes or less from the viewpoint of productivity. Further, in the case of steam, it is thought that a large shrinkage rate can be obtained in a shorter time than with liquid. Contact with the aqueous medium may be performed under normal pressure or under reduced pressure (eg, vacuum).

The method of bringing the modified fibroin filament or the modified fibroin staple into contact with the aqueous medium includes a method of immersing the modified fibroin filament or the modified fibroin staple in the aqueous medium, a method of spraying the modified fibroin filament or the modified fibroin staple with the steam of the aqueous medium, and a method of contacting the modified fibroin filament or the modified fibroin staple with the steam of the aqueous medium. Examples include a method of exposing modified fibroin filaments or modified fibroin staples to the environment. When the aqueous medium is steam, contacting the aqueous medium to the modified fibroin filaments or modified fibroin staples can be performed using common steam set equipment. Specific examples of the steam setting device include devices such as product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and product name: EPS-400 (manufactured by Tsujii Senko Kogyo Co., Ltd.). A specific example of a method for crimping modified fibroin filaments or modified fibroin staples using steam from an aqueous medium is to store modified fibroin filaments or modified fibroin staples in a predetermined storage chamber, and introduce steam from an aqueous medium into the storage chamber. One example of this is to bring the modified fibroin filament or the modified fibroin staple into contact with steam while adjusting the temperature inside the storage chamber to the above-mentioned predetermined temperature (for example, 100° C. to 230° C.).

In the crimping process of modified fibroin filaments or modified fibroin staples by contact with an aqueous medium, preferably no tensile force is applied to the single fibers or bundles (skeins, etc.) of modified fibroin filaments or modified fibroin staples ( This is carried out in a state in which no tension is applied in the fiber axial direction) or in a state in which a predetermined amount of tension is applied (a predetermined amount of tension is applied in the fiber axial direction). By adjusting the tensile force applied to the modified fibroin filament or modified fibroin staple at this time, it is possible to control the degree of crimp. A method for preparing the tensile force to be applied to modified fibroin filaments and modified fibroin staples is, for example, by suspending weights of various weights on modified fibroin filaments and modified fibroin staples. A method of adjusting the load, a method of fixing both ends of the filament and staple in a relaxed state and varying the amount of slack, a method of winding the filament around a body to be wound such as a paper tube or bobbin, and a method of Examples include a method of appropriately changing the winding force (tightening force to the paper tube or bobbin).

After contacting the modified fibroin filament or modified fibroin staple with the aqueous medium, it may be further dried. The drying method is not particularly limited, and the drying may be natural drying or drying with hot air or hot rollers. The drying temperature is not particularly limited, and may be, for example, 20 to 150°C, preferably 40 to 120°C, and more preferably 60 to 100°C.

In addition, from the viewpoint of further increasing the shrinkage rate of the artificial fibroin spun yarn, it is preferable not to carry out the crimping process (particularly the water crimping process). Furthermore, even when the crimping process is carried out, the shrinkage rate of the artificial fibroin spun yarn can be made higher by spinning using a mixture of crimped staples and uncrimped staples in the spinning process. .

The artificial fibroin spun yarn may be a single yarn or a mixed yarn such as a double yarn. Examples of the artificial fibroin spun yarn include a spun yarn of 100% modified fibroin, a blended yarn of 100% modified fibroin staple and at least one selected from other protein staples, chemical fiber staples, etc., and modified fibroin and other yarns. Examples include a spun yarn using a staple containing the following components, and a blended yarn using a staple containing modified fibroin and other components with at least one selected from other protein staples, chemical fiber staples, and the like.

In order to make the modified fibroin staple easier to loosen, an oil agent may be applied in advance to the modified fibroin staple before the spinning process. Oiling can be carried out at any stage in the manufacturing process. For example, the oil may be applied 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 may be any known oil agent used for general purposes such as preventing static electricity, reducing friction, imparting flexibility, imparting water repellency, etc., and imparting functionality. Either of these can be used.

<Artificial fibroin spun yarn>
The artificial fibroin spun yarn according to the present embodiment contains the above-described modified fibroin and is capable of high shrinkage simply by contacting with moisture (water, steam, etc.). The term "high shrinkage possible" means that the shrinkage rate defined by the following formula II is more than 1%.
Shrinkage rate = {1 - (Length of artificial fibroin spun yarn subjected to shrinkage processing including contact with water below the boiling point / Length of artificial fibroin spun yarn before applying the above shrinkage treatment)} x 100 (% )...(Formula II)

The artificial fibroin spun yarn according to the present embodiment contains the above-mentioned modified fibroin and has a shrinkage rate defined by formula II of more than 1%, so it has higher shrinkage than conventionally known protein spun yarns. It can also be shrunk at a higher shrinkage rate under milder conditions than conventionally known spun yarns made of synthetic fibers.

The shrinkage rate of the artificial fibroin spun yarn according to this embodiment is 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, It may be 15% or more, 20% or more, 25% or more, or 30% or more. The upper limit of the shrinkage rate is not particularly limited, but 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, if the artificial fibroin spun yarn according to the present embodiment has no history of contact with moisture (water, water vapor, etc.), including the modified fibroin fiber (meaning the filament after spinning) that is the raw material, High shrinkage is possible. That is, when there is no history of contact with moisture, the shrinkage rate of the artificial fibroin spun yarn according to the present embodiment may be, for example, more than 7%, preferably more than 15%, and more than 25%. is more preferably 32% or more, even more preferably 40% or more, even more preferably 48% or more, particularly preferably 56% or more, and particularly preferably 64% or more. It is especially more preferable that it is, and it is most preferable that it is 72% or more. The upper limit of the shrinkage rate is usually 80% or less.

The artificial fibroin spun yarn according to the present embodiment may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, or 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 may have a maximum hygroscopic heat generation value of more than 0.025° C./g, which is determined according to the following formula A.
Formula A: Maximum hygroscopic exotherm = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then transferred to a high humidity environment) - (The sample temperature is transferred to a high humidity environment) Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (°C) / Sample weight (g)
In Formula A, the low humidity environment means an environment with a temperature of 20° C. and a relative humidity of 40%, and the high humidity environment means an environment with a temperature of 20° C. and a relative humidity of 90%.

The artificial fibroin spun yarn according to the present embodiment may have a maximum hygroscopic heat generation value of 0.026°C/g or more, 0.027°C/g or more, or 0.028°C/g or more. 0.029°C/g or more, 0.030°C/g or more, 0.035°C/g or more, 0.040°C/g It may be more than that. There is no particular restriction on the upper limit of the maximum hygroscopic exotherm, but it is usually 0.060° C./g or less.

The artificial fibroin spun yarn according to the present embodiment may have a heat retention index of 0.20 or more determined according to the following formula B.
Formula B: Heat retention index = heat retention rate (%) / basis weight of sample (g/m ² )

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, or 0.28 or more. , may be 0.30 or more, and may be 0.32 or more. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.

(Shrink processing)
Shrinkage processing includes contacting the artificial fibroin spun yarn with water below the boiling point (hereinafter also referred to as "contact step"). The shrinking process may further include drying the artificial fibroin spun yarn after contact with water (after the contact step) (hereinafter also referred to as "drying step").

The temperature of the water brought into contact with the artificial fibroin spun yarn in the contacting step may be below the boiling point. This improves handling properties, shrinkage workability, and the like. In addition, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit of the water temperature is preferably 10°C or higher, more preferably 40°C or higher, and even more preferably 70°C or higher. . The upper limit of the water temperature is preferably 90°C or less.

In the contacting step, the method of bringing water into contact with the artificial fibroin spun yarn is not particularly limited. Examples of such methods include immersing the artificial fibroin yarn in water, spraying the artificial fibroin yarn with water at room temperature or in a heated steam state, and filling the artificial fibroin yarn with water vapor. For example, exposure to a high-humidity environment may be mentioned. Among these methods, a method in which the artificial fibroin spun yarn is immersed in water is preferred because it can effectively shorten the shrinkage time and simplify the processing equipment in the contact step.

The drying step is a step of drying the artificial fibroin spun yarn that has undergone the contact step. Drying may be, for example, natural drying or forced drying using drying equipment. As the drying equipment, any known contact type or non-contact type drying equipment can be used. Furthermore, the drying temperature is not limited as long as it is lower than the temperature at which proteins (modified fibroin, etc.) contained in the artificial fibroin yarn are decomposed or the artificial fibroin yarn is thermally damaged. Generally, the temperature is in the range of 20 to 150°C, preferably in the range of 50 to 100°C. When the temperature is in this range, the artificial fibroin yarn is dried more quickly and efficiently without thermal damage to the artificial fibroin yarn or decomposition of proteins contained in the artificial fibroin yarn. The drying time is appropriately set depending on the drying temperature and the like, and for example, a time is adopted in which the influence of overdrying on the quality and physical properties of the artificial fibroin spun yarn can be eliminated as much as possible.

The shrinkage rate of the artificial fibroin spun yarn according to this embodiment can be controlled by, for example, controlling the temperature of the water in contact, the contact time with water, and the tensile force when bringing it into contact with water.

<High shrinkage artificial fibroin spun yarn and its manufacturing method>
Since the artificial fibroin spun yarn according to the present embodiment is capable of high shrinkage, a highly shrinkable artificial fibroin spun yarn can be obtained, for example, through the above-mentioned shrinkage processing (contact step and, if necessary, drying step). .

The high shrinkage artificial fibroin spun yarn according to the present embodiment has a shrinkage rate defined by the following formula I of 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)} x 100 (%) ... (Formula I)

The shrinkage rate of the high shrinkage artificial fibroin spun yarn according to this embodiment is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, and 10%. It may be 15% or more, 20% or more, 25% or more, or 30% or more. The upper limit of the shrinkage rate is not particularly limited, but 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, if the artificial fibroin spun yarn according to the present embodiment has no history of contact with moisture (water, water vapor, etc.), including the modified fibroin fiber (meaning the filament after spinning) that is the raw material, High shrinkage is possible. That is, when there is no history of contact with moisture, the shrinkage rate of the high-shrinkage artificial fibroin spun yarn according to the present embodiment may be, for example, more than 7%, preferably more than 15%, and more than 25%. It is more preferably at least 32%, even more preferably at least 40%, even more preferably at least 48%, particularly preferably at least 56%. % or more is especially more preferable, and most preferably 72% or more. The upper limit of the shrinkage rate is usually 80% or less.

The high shrinkage artificial fibroin spun yarn according to the present embodiment has a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more. It may be 26 or more, 28 or more, 29 or more, or 30 or more.

The high-shrinkage artificial fibroin spun yarn according to the present embodiment may have a maximum hygroscopic heat generation value of more than 0.025° C./g, which is determined according to the following formula A.
Formula A: Maximum hygroscopic exotherm = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then transferred to a high humidity environment) - (The sample temperature is transferred to a high humidity environment) Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (°C) / Sample weight (g)
In Formula A, the low humidity environment means an environment with a temperature of 20° C. and a relative humidity of 40%, and the high humidity environment means an environment with a temperature of 20° C. and a relative humidity of 90%.

The high shrinkage artificial fibroin spun yarn according to the present embodiment may have a maximum hygroscopic exotherm of 0.026°C/g or more, 0.027°C/g or more, and 0.028°C/g. or more, may be 0.029°C/g or more, may be 0.030°C/g or more, may be 0.035°C/g or more, and may be 0.040°C /g or more. There is no particular restriction on the upper limit of the maximum hygroscopic exotherm, but it is usually 0.060° C./g or less.

The high-shrinkage artificial fibroin spun yarn according to the present embodiment may have a heat retention index of 0.20 or more determined according to formula B below.
Formula B: Heat retention index = heat retention rate (%) / basis weight of sample (g/m ² )

The heat retention index of the high shrinkage 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. It may be 0.30 or more, and it may be 0.32 or more. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.

[Method for shrinking artificial fibroin spun yarn]
The method for shrinking an artificial fibroin spun yarn according to the present embodiment includes a step of bringing an artificial fibroin spun yarn containing modified fibroin into contact with water below the boiling point and causing the artificial fibroin yarn to shrink. The shrinking step may further include drying the artificial fibroin yarn after contact with water. That is, the method for shrinking the artificial fibroin spun yarn according to this embodiment can be carried out in the same manner as the shrinking process described above.

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

[Test Example 1: Production of modified fibroin (modified spider silk fibroin)]
(1) Preparation of modified fibroin expression vector Modified fibroin having the amino acid sequence shown by SEQ ID NO: 18 (PRT399), modified fibroin having the amino acid sequence shown by SEQ ID NO: 12 (PRT380), and amino acid sequence shown by SEQ ID NO: 13. A modified fibroin (PRT410) having the amino acid sequence shown in SEQ ID NO: 15 (PRT799), and a modified fibroin (PRT918) having the amino acid sequence shown in SEQ ID NO: 37 were designed.

Nucleic acids encoding proteins having the designed amino acid sequences were synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the stop codon. These five types of nucleic acids were cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was treated with restriction enzymes NdeI and EcoRI to cut it out, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

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

The seed culture solution was added to a jar fermenter supplemented with 500 ml of production medium (Table 5) so that the OD ₆₀₀ was 0.05. Culture was carried out by keeping the culture solution temperature at 37° C. and controlling the pH to be constant at 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

(3) Purification of modified fibrons The bacterial cells collected 2 hours after adding IPTG were washed with 20mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20mM Tris-HCl buffer (pH 7.4) containing about 1mM PMSF, and the cells were disrupted using a high-pressure homogenizer (GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH 7.0) to a concentration of 100mg/mL and incubated at 60°C. The mixture was stirred with a stirrer for 30 minutes to dissolve the mixture. After dissolution, dialysis was performed against water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation. Water was removed from the collected aggregated proteins using a freeze dryer to obtain freeze-dried powders of the desired modified fibroin (PRT399, PRT380, PRT410, PRT799 and PRT918).

[Reference Example 1: Production and shrinkage evaluation of modified fibroin fiber (filament)]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and the lyophilized powder of modified fibroin (PRT399, PRT380, PRT410 or PRT799) obtained in Test Example 1 was added thereto. It was added to a concentration of 18% by mass or 24% by mass (see Table 6), and dissolved for 3 hours using a shaker. Thereafter, insoluble matters 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 spun and drawn modified fibroin fibers (filaments) were produced by dry-wet spinning using a spinning device similar to the spinning device 10 shown in FIG. The spinning apparatus used is a spinning apparatus 10 shown in FIG. 6, which has a second undrawn yarn manufacturing apparatus ( (second bath). The conditions for wet-dry spinning are as follows.
Extrusion nozzle diameter: 0.2mm
Liquid and temperature in 1st to 3rd baths: See Table 6 Total stretching ratio: See Table 6 Drying temperature: 60°C

(Contractability evaluation)
The shrinkage rate of each modified fibroin fiber (filament) obtained in Production Examples 1 to 19 was evaluated. That is, the shrinkage rate (hereinafter sometimes referred to as "primary shrinkage rate") when each modified fibroin fiber is subjected to shrinkage processing 1 consisting of contacting it with water below the boiling point, and the water below the boiling point. The shrinkage rate (hereinafter sometimes referred to as "secondary shrinkage rate") was evaluated when the shrinkage process 2 was performed, which consisted of contacting the sample with the material and then drying it at room temperature.

<Primary shrinkage rate>
A plurality of modified fibroin fibers for testing each having a length of 30 cm were cut out from the rolled modified fibroin fibers obtained in Production Examples 1 to 19. A modified fibroin fiber bundle having a fineness of 150 denier was obtained by bundling these modified fibroin fibers. A 0.8 g lead weight was attached to each modified fibroin fiber bundle, and in that state, each modified fibroin fiber bundle was immersed in water at the temperatures shown in Tables 7 to 10 for 10 minutes (shrinkage processing 1). Thereafter, the length of each modified fibroin fiber bundle was measured in water. The length measurement of the modified fibroin fiber bundle in water was carried out with a 0.8 g lead weight attached to the modified fibroin fiber bundle in order to eliminate curling of the modified fibroin fiber bundle. Next, the primary shrinkage rate (%) of each modified fibroin fiber was calculated according to the following formula III. In formula III, L0 indicates the length of the modified fibroin fiber bundle before shrinkage processing (here, 30 cm), and Lw indicates the length of the modified fibroin fiber bundle after shrinkage processing 1.
Formula III: Shrinkage rate (primary shrinkage rate) = {1-(Lw/L0)} x 100 (%)

<Secondary shrinkage rate>
After immersion in water for evaluation of primary shrinkage, the modified fibroin fiber bundles were removed from the water. The taken-out modified fibroin fiber bundle was left to dry at room temperature for 2 hours with a 0.8 g lead weight attached (shrinking process 2). After drying, the length of each modified fibroin fiber bundle was measured. Next, the secondary shrinkage rate (%) of each modified fibroin fiber was calculated according to the following formula IV. In formula IV, L0 indicates the length of the modified fibroin fiber bundle (30 cm here) before shrinkage processing, and Lwd indicates the length of the modified fibroin fiber bundle after shrinkage processing 2.
Formula IV: Shrinkage rate (secondary shrinkage rate) = {1-(Lwd/L0)} x 100 (%)

The results are shown in Tables 7-10.

From the above results, it can be seen that the modified fibroin fiber (filament) according to the present invention has a sufficiently high shrinkage rate. Therefore, it is recognized that the artificial fibroin spun yarn spun from the modified fibroin fiber (filament) according to the present invention also has a sufficiently high shrinkage rate. In addition, the shrinkage rate of the modified fibroin fibers (filaments) and artificial fibroin spun yarns according to the present invention can be controlled, for example, by controlling the temperature of the water they are brought into contact with, the time they are in contact with the water, and the tensile force when they are brought into contact with the water. I also understand that it is possible.

[Example 1: Production and evaluation of artificial fibroin spun yarn]
(1) Production of modified fibroin fiber (filament) Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and the modified fibroin (PRT799) obtained in Test Example 1 was added to it as a solvent. Freeze-dried powder was added to a concentration of 24% by mass and dissolved for 3 hours using a shaker. Thereafter, insoluble matters 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 filament was wound onto a bobbin. The conditions for wet-dry spinning are as follows.
Temperature of coagulation liquid (methanol): 5-10℃
Stretching ratio: 5 times Drying temperature: 80℃

(2) Manufacture of artificial fibroin spun yarn Modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm using a tabletop fiber cutting machine to obtain modified fibroin staples. A portion of the obtained staple was crimped by immersing it in water at 40°C for 1 minute and then dried at 40°C for 18 hours to obtain a crimped staple. Next, staples containing crimped staples and uncrimped staples at a ratio of 7:3 (weight ratio) were spun using a known spinning device to obtain artificial fibroin spun yarn 1. The fineness (count) of the artificial fibroin spun yarn 1 was 30 Nm.

(3) Evaluation of artificial fibroin spun yarn (water shrinkage test)
Artificial fibroin spun yarn 1 was cut into a length of 60 cm, immersed in water at 40° C. for 1 minute, and then air-dried. The length of the spun yarn after air drying was measured, and the shrinkage rate was determined according to formula V below. The results are shown in Table 11.
Shrinkage rate = {1 - (Length of spun yarn after air drying/Length of spun yarn before immersion in water)} x 100 (%) ... (Formula V)
Note that "the length of the spun yarn before being immersed in water" is 60 cm.

[Example 2: Production and evaluation of artificial fibroin spun yarn]
(1) Production of modified fibroin fiber (filament) Modified fibroin filament was obtained in the same manner as in Example 1.

(2) Manufacture of artificial fibroin spun yarn Modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm using a tabletop fiber cutting machine to obtain modified fibroin staples. The obtained staple (uncrimped staple) was spun using a known spinning device to obtain an artificial fibroin spun yarn 2. The fineness (count) 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 manner as in Example 1. The results are shown in Table 11.

[Comparative Example 1: Production and evaluation of artificial fibroin spun yarn]
(1) Production of modified fibroin fiber (filament) Instead of the lyophilized powder of modified fibroin (PRT799), the lyophilized powder of modified fibroin (PRT918) obtained in Test Example 1 and the lyophilized powder of modified fibroin (PRT799) were used. A modified fibroin filament was produced in the same manner as in Example 1, except that a freeze-dried powder containing 6:4 (weight ratio) was used.

(2) Manufacture of artificial fibroin spun yarn Modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm using a tabletop fiber cutting machine to obtain modified fibroin staples. The obtained staples were crimped by immersing them in water at 40° C. for 1 minute and then dried at 40° C. for 18 hours to obtain crimped staples. Next, the crimped staple was spun using a known spinning device to obtain an artificial fibroin spun yarn 3. The fineness (count) 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 manner as in Example 1. The results are shown in Table 11.

[Comparative Example 2: Evaluation of PET spun yarn]
For comparison, a commercially available single spun yarn (PET spun yarn) made of 100% PET and having a fineness of 30 Nm was purchased. A water shrinkage test was conducted in the same manner as in Example 1 to determine the shrinkage rate of the PET spun yarn. The results are shown in Table 11.

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 texture and flexibility. In addition, the high shrinkage artificial fibroin spun yarn of the present invention is safe because it can be produced by contacting the artificial fibroin spun yarn with water below the boiling point and optionally drying after contact with water. production is possible.

[Test Example 2: Production of modified fibroin]
Designed modified fibroin (PRT918) having the amino acid sequence shown by SEQ ID NO: 37, modified fibroin (PRT966) having the amino acid sequence shown by SEQ ID NO: 40, and modified fibroin (PRT799) having the amino acid sequence shown by SEQ ID NO: 15. did. A nucleic acid encoding the designed modified fibroin was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

E. coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 12) containing ampicillin so that the OD ₆₀₀ was 0.005. The culture solution temperature was maintained at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter supplemented with 500 mL of production medium (Table 13) so that the OD ₆₀₀ was 0.05. Culture was carried out by keeping the culture solution temperature at 37° C. and controlling the pH to be constant at 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

The bacterial cells collected 2 hours after adding IPTG were washed with 20mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted using a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH 7.0) to a concentration of 100mg/mL and incubated at 60°C. The mixture was stirred with a stirrer for 30 minutes to dissolve the mixture. After dissolution, dialysis was performed against water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). Modified fibroin (PRT918, PRT966, and PRT799) was obtained by collecting the white aggregated protein obtained after dialysis by centrifugation, removing water with a freeze dryer, and collecting freeze-dried powder.

PRT918 and PRT966 are hydrophobically modified fibroins with average HI greater than zero. PRT799 is a hydrophilic modified fibroin with an average HI of 0 or less.

[Reference example 2: Flame retardant evaluation of modified fibroin]
Dimethyl sulfoxide (DMSO) in which LiCl is dissolved at a concentration of 4.0% by mass is prepared as a solvent, and the freeze-dried powder of modified fibroin (PRT799) obtained in Test Example 2 is added thereto to a concentration of 24% by mass. and dissolved using a shaker for 3 hours. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution was filtered at 90°C through a metal filter with an opening of 5 μm, then allowed to stand in a 30 mL stainless steel syringe to defoam, and then coagulated with 100% methanol from a solid nozzle with a needle diameter of 0.2 mm. I vomited it into the bathtub. The discharge temperature was 90°C. After coagulation, the obtained yarn was wound up and naturally dried to obtain a modified fibroin fiber (raw material fiber).

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

The flammability test was conducted in accordance with the test method for synthetic resins in the form of powder or granules or with a low melting point published by the Chief of the Hazardous Materials Control Division of the Fire and Disaster Management Agency, Fire Safety No. 50, May 31, 1995. The test was conducted under conditions of a temperature of 22° C., a relative humidity of 45%, and an atmospheric pressure of 1021 hPa. The measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)) are shown in Table 14.

As a result of the flammability test, the limiting oxygen index (LOI) value of the knitted fabric made of modified fibroin (PRT799) fiber was 27.2. Generally, if the LOI value is 26 or more, it is considered to be flame retardant. It can be seen that the modified fibroin has excellent flame retardancy.

[Reference Example 3: Evaluation of hygroscopic exothermic property of modified fibroin]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0% by mass was prepared as a solvent, and the freeze-dried powder of modified fibroin obtained in Test Example 2 was added thereto at a concentration of 24% by mass. , and dissolved for 3 hours using a shaker. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution was filtered at 60°C through a metal filter with an opening of 5 μm, then left to stand in a 30 mL stainless steel syringe to defoam, and then coagulated with 100% methanol from a solid nozzle with a needle diameter of 0.2 mm. I vomited it into the bathtub. The discharge temperature was 60°C. After coagulation, the obtained yarn was wound up and naturally dried to obtain a modified fibroin fiber (raw material fiber).

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

Using each raw material fiber, a knitted fabric was manufactured by flat knitting using a flat knitting machine. The knitted fabric using PRT918 fiber as the raw material fiber had a thickness of 1/30N (single thread yarn) and a gauge number of 18. The knitted fabric using PRT799 fiber as the raw material fiber had a thickness of 1/30N (fleece count single yarn) and a gauge number of 16. The thickness and gauge number of the knitted fabrics using other raw material fibers were adjusted so that they had almost the same cover factor as the knitted fabrics using PRT918 fibers and PRT799 fibers. Specifically, it is as follows.
Wool Thickness: 2/30N (double yarn), Gauge number: 14
Cotton Thickness: 2/34N (double thread), Gauge number: 14
Tencel Thickness: 2/30N (double thread), Gauge number: 15
Rayon thickness: 1/38N (single yarn), gauge number: 14
Polyester Thickness: 1/60N (single thread), Gauge number: 14

Two pieces of knitted fabric cut to 10 cm x 10 cm were put together and the four sides were sewn together to prepare a test piece (sample). After leaving the test piece in a low humidity environment (temperature 20 ± 2 °C, relative humidity 40 ± 5%) for more than 4 hours, it was transferred to a high humidity environment (temperature 20 ± 2 °C, relative humidity 90 ± 5%). The temperature was measured at 1 minute intervals for 30 minutes using a temperature sensor attached to the center of the interior.

From the measurement results, the maximum hygroscopic exotherm was determined according to formula A below.
Formula A: Maximum hygroscopic exotherm = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then transferred to a high humidity environment) - (The sample temperature is transferred to a high humidity environment) Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (°C) / Sample weight (g)

FIG. 7 is a graph showing an example of the results of the moisture absorption heat generation test. The horizontal axis of the graph indicates the time (minutes) in which the sample was left in the high humidity environment, with the time point at which the sample was transferred from a low humidity environment to a high humidity environment being 0. The vertical axis of the graph indicates the temperature measured by the temperature sensor (sample temperature). In the graph shown in FIG. 7, the point marked M corresponds to the highest value of the sample temperature.

Table 15 shows the calculation results of the maximum hygroscopic heat generation.

As shown in Table 15, it can be seen that the modified fibroin (PRT918 and PRT799) has a higher maximum hygroscopic heat generation value and is superior in hygroscopic heat generation property compared to existing materials.

[Reference example 4: Heat retention evaluation of modified fibroin]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0% by mass was prepared as a solvent, and the freeze-dried powder of modified fibroin obtained in Test Example 2 was added thereto at a concentration of 24% by mass. , and dissolved for 3 hours using a shaker. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution was filtered at 60°C through a metal filter with an opening of 5 μm, then left to stand in a 30 mL stainless steel syringe to defoam, and then coagulated with 100% methanol from a solid nozzle with a needle diameter of 0.2 mm. I vomited it into the bathtub. The discharge temperature was 60°C. After coagulation, the obtained yarn was wound up and naturally dried to obtain a modified fibroin fiber (raw material fiber).

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

Using each raw material fiber, a knitted fabric was manufactured by flat knitting using a flat knitting machine. The knitted fabric using PRT966 fiber as the raw material fiber had a count of 30 Nm, number of twists: 1, number of gauges: 18 GG, and basis weight: 90.1 g/m ² . The knitted fabric using PRT799 fiber as the raw material fiber had a count of 30 Nm, number of twists: 1, gauge number GG: 16, and basis weight: 111.0 g/m ² . The thickness and gauge number of the knitted fabrics using other raw material fibers were adjusted so that they had almost the same cover factor as the knitted fabrics using PRT966 fibers and PRT799 fibers. Specifically, it is as follows.
Wool count: 30Nm, number of twists: 2, number of gauges: 14GG, basis weight: 242.6g/m ²
Silk count: 60Nm, number of twists: 2, number of gauge: 14GG, basis weight: 225.2g/m ²
Cotton count: 34Nm, number of twists: 2, number of gauge: 14GG, basis weight: 194.1g/m ²
Rayon count: 38Nm, number of twists: 1, number of gauges: 14GG, basis weight: 181.8g/m ²
Polyester count: 60Nm, number of twists: 1, number of gauges: 14GG, basis weight: 184.7g/ ^m2

Heat retention was evaluated using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd. using a dry contact method (a method assuming that the skin and clothing come into direct contact in a dry state). One piece of knitted fabric cut to 20 cm x 20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30° C.), and the amount of heat (a) dissipated through the test piece was determined under conditions of a wind speed of 30 cm/sec in the wind tunnel. The amount of heat dissipated (b) 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) x 100

From the measurement results, the heat retention index was determined according to formula B below.
Formula B: Heat retention index = heat retention rate (%) / basis weight of sample (g/m ² )

Table 16 shows the calculation results of the heat retention index. The higher the heat retention index, the better the material can be evaluated for heat retention.

As shown in Table 16, it can be seen that the modified fibroin (PRT966 and PRT799) has a higher heat retention index and is superior in heat retention than existing materials.

1... Extrusion device, 2... Undrawn yarn manufacturing device, 3... Moist heat stretching device, 4... Drying device, 6... Dope liquid, 10... Spinning device, 20... Coagulation liquid bath, 21... Stretching bath, 36... Modified fibroin fiber (filament).

Claims (17)

A shrunken artificial fibroin yarn comprising modified fibroin, the yarn comprising:
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif,
The domain sequence is different from the amino acid sequence of naturally occurring fibroin,
A high shrinkage artificial fibroin spun yarn having a shrinkage rate defined by the following formula I of 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)} x 100 (%) ... (Formula I)
[In Formula 1 and Formula 2, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% of the total number of amino acid residues in (A) _n motif. That's all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The high shrinkage artificial fibroin spun yarn according to claim 1, wherein the modified fibroin is modified spider silk fibroin. A step of bringing an artificial fibroin spun yarn containing modified fibroin into contact with water below the boiling point to cause it to shrink,
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif,
The domain sequence is different from the amino acid sequence of naturally occurring fibroin,
A method for producing a highly shrinkable artificial fibroin spun yarn, which has a shrinkage rate defined by the following formula I of 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)} x 100 (%) ... (Formula I)
[In Formula 1 and Formula 2, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% of the total number of amino acid residues in (A) _n motif. That's all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The method for producing a high-shrinkage artificial fibroin spun yarn according to claim 3 , wherein the modified fibroin is modified spider silk fibroin. The method for producing a high-shrinkage artificial fibroin spun yarn according to claim 3 or 4 , wherein the temperature of the water is 10 to 90°C. The method for producing a highly shrinkable artificial fibroin spun yarn according to any one of claims 3 to 5 , wherein the shrinking step further includes drying the artificial fibroin spun yarn after contact with the water. A step of bringing an artificial fibroin spun yarn containing modified fibroin into contact with water below the boiling point to cause it to shrink,
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif,
The domain sequence is different from the amino acid sequence of naturally occurring fibroin,
A method for shrinking an artificial fibroin spun yarn, wherein 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)} x 100 (%) ... (Formula I)
[In Formula 1 and Formula 2, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% of the total number of amino acid residues in (A) _n motif. That's all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The method for shrinking artificial fibroin spun yarn according to claim 7 , wherein the modified fibroin is modified spider silk fibroin. The method for shrinking artificial fibroin spun yarn according to claim 7 or 8 , wherein the temperature of the water is 10 to 90°C. The method for shrinking an artificial fibroin yarn according to any one of claims 7 to 9 , wherein the shrinking step further includes drying the artificial fibroin yarn after contact with the water. Contains modified fibroin and has a shrinkage rate defined by the following formula II of more than 1%,
The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif,
An artificial fibroin yarn, wherein the domain sequence is different from the amino acid sequence of naturally-derived fibroin .
Shrinkage rate = {1 - (Length of artificial fibroin spun yarn subjected to shrinkage processing including contact with water below the boiling point / Length of artificial fibroin spun yarn before applying the shrinkage process)} x 100 (% )...(Formula II)
[In Formula 1 and Formula 2, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% of the total number of amino acid residues in (A) _n motif. That's all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
The domain sequence corresponds to the deletion of at least one or more (A) _n motifs compared to naturally occurring fibroin, and has an amino acid sequence with a reduced content of (A) _n motifs. The high shrinkage artificial fibroin spun yarn according to claim 1 or 2 .
[In formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
The domain sequence has a reduced content of glycine residues, which corresponds to substitution of at least one or more glycine residues in REP with another amino acid residue, compared to naturally occurring fibroin. The highly contractile artificial fibroin spun yarn according to claim 1 or 2 , which has an amino acid sequence.
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% or more of the total number of amino acid residues in (A) _n motif. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m ,
The domain sequence has one or more amino acid residues in REP replaced with amino acid residues having a larger hydrophobicity index than naturally occurring fibroin, and/or one or more hydrophobic residues in REP. The highly contractile artificial fibroin spun yarn according to claim 1 or 2 , which has an amino acid sequence that includes a region with a locally large hydrophobicity index, which corresponds to insertion of an amino acid residue with a large sex index.
[In Formula 1, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% or more of the total number of amino acid residues in (A) _n chief. . REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The modified fibroin comprises a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m - (A) _n motif,
The content of glutamine residues in the domain sequence corresponds to deletion of one or more glutamine residues in REP or substitution with other amino acid residues as compared to naturally occurring fibroin. The high-shrinkage artificial fibroin spun yarn according to claim 1 or 2 , which has an amino acid sequence with a reduced number of amino acids.
[In Formula 1 and Formula 2, (A) _n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and the number of alanine residues is 40% of the total number of amino acid residues in (A) _n motif. That's all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or may have different amino acid sequences. A plurality of REPs may have the same or different amino acid sequences. ] The highly contractile artificial fibroin spun yarn according to claim 1 or 2 , wherein the modified fibroin has a limiting oxygen index (LOI) value of 26.0 or more. The highly contractible artificial fibroin spun yarn according to claim 1 or 2 , wherein the modified fibroin has a maximum hygroscopic exotherm determined according to the following formula A of more than 0.025° C./g.
Formula A: Maximum hygroscopic exotherm = {(The maximum value of the sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then transferred to a high humidity environment) - (The sample temperature is Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (°C) / Sample weight (g)
[In Formula A, the low humidity environment means an environment with a temperature of 20° C. and a relative humidity of 40%, and the high humidity environment means an environment with a temperature of 20° C. and a relative humidity of 90%. ]

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