JP2021152224A - High-density unwoven fabric and method for manufacturing the same - Google Patents

Abstract

To provide a method for manufacturing a high-density unwoven fabric including a protein fiber.SOLUTION: A method for manufacturing a high-density unwoven fabric includes a contraction step for bringing a raw material unwoven fabric formed from fibers including a protein fiber at least in a part into contact with moisture and contracting the fabric.SELECTED DRAWING: None

Description

The present invention relates to a high-density non-woven fabric and a method for producing a high-density non-woven fabric.

Nonwoven fabrics are widely used as clothing products, medical / hygiene products, various industrial products, and the like. The non-woven fabric may be required to have a so-called high density, in which the fiber density is increased so as to reduce the gaps between the fibers. For example, in clothing products and the like, high density may be required in order to improve windproofness or dustproofness. Further, in a filter or the like, which is a kind of industrial product, high density may be required in order to improve the collectability.

When obtaining a high-density non-woven fabric, a method of tightly entwining fine threads is generally adopted, but this method has limitations. Therefore, for example, Patent Document 1 and the like propose a method of obtaining a high-density non-woven fabric by needle punching while heating a non-woven fabric web made of synthetic fibers such as polyethylene terephthalate fiber, polyamide fiber and polyurethane fiber. ..

Japanese Unexamined Patent Publication No. 9-87951 etc.

However, the fact is that the non-woven fabric containing protein fibers has not been sufficiently studied.

An object of the present invention is to provide a method for producing a high-density non-woven fabric containing protein fibers. It is also an object of the present invention to provide a high density non-woven fabric containing protein fibers.

The present invention relates to, for example, the following inventions.
[1]
A method for producing a high-density non-woven fabric, comprising a shrinkage step of bringing a raw non-woven fabric formed of fibers containing at least a part of protein fibers into contact with moisture to shrink the raw material non-woven fabric.
[2]
The production method according to [1], wherein the fiber density increase rate defined by the following formula I is 20% or more.
Fiber density increase rate = {(fiber density of high-density non-woven fabric / fiber density of raw non-woven fabric) -1} x 100 (%) ... (Formula I)
[3]
The production method according to [1] or [2], wherein the protein fiber has a shrinkage rate at wet time of 2% or more as defined by the following formula II.
Shrinkage rate when wet = {1- (length of protein fiber moistened by contact with moisture / length of protein fiber before contact with moisture)} × 100 (%)… (Formula II)
[4]
The production method according to any one of [1] to [3], wherein the protein fiber has a shrinkage rate at drying of more than 7% as defined by the following formula III.
Shrinkage rate during drying = {1- (length of protein fiber dried after contact with moisture / wet state / length of protein fiber before contact with moisture)} × 100 (%)… (formula III)
[5]
The production method according to any one of [1] to [4], wherein the temperature of the water is lower than the boiling point.
[6]
Further provided with a forming process for forming the raw material non-woven fabric,
The production method according to any one of [1] to [5], wherein the forming step includes adjusting the fiber density of the raw material nonwoven fabric.
[7]
The production method according to any one of [1] to [6], wherein the protein fiber is a fibroin fiber.
[8]
The production method according to any one of [1] to [7], wherein the protein fiber is a modified spider silk fibroin fiber.
[9]
A high-density non-woven fabric having fibers containing at least a part of protein fibers and the fibers being water-shrinked.
[10]
A high-density non-woven fabric having fibers containing at least a part of protein fibers and being water-shrinked.
[11]
The high-density nonwoven fabric according to [10], wherein the fiber density increase rate defined by the following formula IV is 20% or more.
Fiber density increase rate = {(fiber density of high-density non-woven fabric after water shrinkage / fiber density of non-woven fabric before water shrinkage) -1} x 100 (%) ... (Equation IV)
[12]
It has fibers that contain at least a portion of protein fibers and
A high-density non-woven fabric in which the fiber density exceeds the maximum value of the fiber density that can be achieved with the non-woven fabric formed from the above fibers.
[13]
The high-density non-woven fabric according to any one of [9] to [12], wherein the protein fiber is a fibroin fiber.
[14]
The high-density non-woven fabric according to any one of [9] to [13], wherein the protein fiber is a modified spider silk fibroin fiber.

According to the present invention, it is possible to provide a method for producing a high-density non-woven fabric containing protein fibers. According to the present invention, it is also possible to provide a high-density non-woven fabric containing protein fibers.

It is a schematic diagram which shows the domain sequence of the modified fibroin which concerns on one Embodiment. It is a figure which shows the distribution of the value of z / w (%) of naturally-derived fibroin. It is a figure which shows the distribution of the value of x / y (%) of naturally-derived fibroin. It is a schematic diagram which shows the domain sequence of the modified fibroin which concerns on one Embodiment. It is a schematic diagram which shows the domain sequence of the modified fibroin which concerns on one Embodiment. It is explanatory drawing which shows typically an example of the spinning apparatus for manufacturing the modified fibroin fiber (filament).

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

[Manufacturing method of high-density non-woven fabric]
The method for producing a high-density non-woven fabric according to the present embodiment includes a shrinkage step of bringing a raw non-woven fabric formed of fibers containing at least a part of protein fibers into contact with water to shrink the non-woven fabric. The method for producing a high-density non-woven fabric according to the present embodiment may further include a forming step for forming a raw material non-woven fabric.

(Formation process)
<Protein>
The protein may be, for example, a structural protein and a modified structural protein derived from the structural protein. Structural protein means a protein that forms or retains its structure, morphology, etc. in vivo. Examples of the structural protein include fibroin, collagen, resilin, elastin, keratin and the like.

The fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin (spider silk protein), and hornet silk fibroin. The fibroin is preferably spider silk fibroin (spider silk protein). Spider silk fibroin includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. Examples of the natural spider silk fibroin include spider silk proteins produced by spiders.

Fibroin is, for example, a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} You may. Fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.

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

The "modified fibroin" may be one in which the amino acid sequence of naturally-derived fibroin is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived). It may be a product in which the amino acid sequence is modified by modifying the gene sequence of fibroin, or an artificially designed and synthesized product (for example, a nucleic acid encoding the designed amino acid sequence) regardless of naturally occurring fibroin. It may have a desired amino acid sequence by chemical synthesis).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically _{corresponding to (A) n} motif of amino acid sequence) and an amorphous region (typically, REP of amino acid sequence). An amino acid sequence that produces (corresponding to.)), _{Which is represented by the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, the (A) _n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2-27. (A) The _number of amino acid residues of the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (A) _{the ratio of the number of alanine residues to the total number of amino acid residues in the n} motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). A plurality of (A) _n motifs present in the domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

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

Naturally-derived fibroin is a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} Yes, specifically, for example, fibroin produced by insects or spiders.

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

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

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

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleic acid sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleic acid sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank sequence No. AAC4011) (Nucleotide sequence) ), U37520 (nucleic acid sequence)), major amplifier spidroin 1 [derived from Latrodictus protein] (GenBank accession number ABR68856 (nucleic acid sequence), EF595246 (nucleic acid sequence)), dragline silk protein (derived from nuclear protein) Numbers AAL32472 (nucleic acid sequence), AF441245 (nucleic acid sequence)), major aminos protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (nucleotide sequence), AJ973155 (nucleotide sequence)), and major protein (GenBank accession number CAM32249.1 (nucleic acid sequence), AM490169 (nucleic acid sequence)), minor amplify silk protein 1 [Nephila proteins] (GenBank accession number AAC14589.1 (nucleotide sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nefilengys cruisetata] (GenBank accession) The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Next, for each pattern, the number of amino acid residues of each REP in _{two adjacent [(A) n motif-REP] units selected is compared.} The comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparing the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), when the 4th REP having a smaller number of amino acid residues is 1, the 5th REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is 1.8 to 11.3 [(A) _n motif-REP] unit set. Shown by solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 _{. The set of [(A) n} motif-REP] units is indicated by a broken line. Indicated.

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

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

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

When the third modified fibroin is a modified fibroin in which _{at least 7 of the (A) n} motifs present in the domain sequence are composed only of alanine residues, the x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, 70% or more is even more preferable, and 80% or more. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

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

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

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

As a more specific example of the third modified fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met) -Contains an amino acid sequence represented by PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Modified fibroin can be mentioned.

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

The x / y value of the amino acid sequence shown in 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 the amino acid sequence shown in SEQ ID NO: 17 and the amino acid sequence shown in SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. 1% and 70.0%.

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

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

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _{The amino acid residue of two adjacent [(A) n} motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3) When x is the maximum value of the total value obtained by adding the numbers of groups and y is the total number of amino acid residues in the domain sequence, x / y is preferably 64.2% or more.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In the present specification, the "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n- motif-REP] _m , or Formula 2: [(A) _n- motif-REP] _m- (A) _{Fibroin containing a domain sequence represented by n-} motif (modified fibroin or naturally derived) In (fibroin), the number of GPGXX motifs contained in the region in all REPs included in the sequence excluding the sequence from _{the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. Let s be the number obtained by multiplying the total number by 3 (that is, corresponding to the total number of G and P in the GPGXX motif), and the _{sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence is taken from the domain sequence. The GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluding (A) _{n motifs.}

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

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

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

In the present specification, the "glutamine residue content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _{fibroin containing a domain sequence represented by n} motif (modified fibroin or naturally derived fibroin) _{In fibroin), all the sequences from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In the REP, the total number of glutamine residues contained in the region is u, and the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) _n. The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif. In the calculation of the glutamine residue content, the _{reason why "the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

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

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

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

The sixth modified fibroin has a REP hydrophobicity of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present specification, the "hydrophobicity of REP" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _{fibroin containing a domain sequence represented by n} motif (modified fibroin or naturally derived fibroin) _{In fibroin), all the sequences from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In the REP of, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the _{n motif.} In the calculation of the hydrophobicity of REP, the _{reason for targeting "the sequence obtained by excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the above-mentioned reason. The same is true.

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

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

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

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

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

In the amino acid sequence shown by SEQ ID NO: 32, all the QQs in the sequence in which the regions of the 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) are repeated twice are replaced with VF. And the remaining Q is replaced with I.

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

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

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

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

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

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

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

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

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

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

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

As a collagen-derived protein, for example, a protein _{containing a domain sequence represented by the formula 3: [REP2] i} (where i in formula 3 represents an integer of 5 to 300. REP2 is Gly-XY. Indicates an amino acid sequence composed of, and X and Y indicate arbitrary amino acid residues other than Gly. Multiple REP2s may have the same amino acid sequence or different amino acid sequences.) .. Specifically, a protein containing the amino acid sequence (Collagen-type4-Kai) shown in SEQ ID NO: 41 can be mentioned. The amino acid sequence shown in SEQ ID NO: 41 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (NCBI GenBank accession number: CAA5635.1, GI: 3702452) obtained from the NCBI database. The amino acid sequence (His tag sequence and hinge sequence) shown in SEQ ID NO: 11 is added to the N-terminal of the amino acid sequence from the 301st residue to the 540th residue.

As a protein derived from resilin, for example, a protein _{containing a domain sequence represented by the formula 4: [REP3] j} (where, in formula 4, j represents an integer of 4 to 300. REP3 is Ser-JJ-. The amino acid sequence composed of Tyr-Gly-U-Pro is shown. J indicates an arbitrary amino acid residue, and it is particularly preferable that it is an amino acid residue selected from the group consisting of Asp, Ser and Thr. U is arbitrary. It is preferable that it is an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser. The plurality of REP4s may have the same amino acid sequence or different amino acid sequences. ) Can be mentioned. Specifically, a protein containing the amino acid sequence (Resilin-Kai) shown in SEQ ID NO: 42 can be mentioned. The amino acid sequence shown in SEQ ID NO: 42 replaces Thr at residue 87 with Ser and Asn at residue 95 in the amino acid sequence of recillin (Axion No. NP 61157, Gl: 24654243 of NCBI GenBank). The amino acid sequence shown by SEQ ID NO: 11 (His tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced with Asp.

Examples of elastin-derived proteins include proteins having amino acid sequences such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, a protein containing the amino acid sequence (elastin short) shown in SEQ ID NO: 43 can be mentioned. The amino acid sequence shown by SEQ ID NO: 43 is the amino acid sequence shown by SEQ ID NO: 11 at the N-terminal of the amino acid sequence from the 121st residue to the 390th residue of the amino acid sequence of accession number AAC98395 of GenBank of NCBI (His tag). Arrangement and hinge arrangement) are added.

Examples of the keratin-derived protein include Type I keratin of Capra hilcus. Specifically, a protein containing the amino acid sequence shown by SEQ ID NO: 44 (amino acid sequence of accession number ACY30466 of GenBank of NCBI: type I keratin 26) can be mentioned.

The above-mentioned structural protein and the modified structural protein derived from the structural protein may be used alone or in combination of two or more.

<Protein manufacturing method>
The protein is expressed, for example, by expressing the nucleic acid in a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced.

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

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

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector that can be autonomously replicated in a host cell or can be integrated into the chromosome of the host and contains a promoter at a position where a nucleic acid encoding a protein can be transcribed is preferably used.

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

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

When a prokaryote is used as a host, as a vector into which a nucleic acid encoding a protein is introduced, for example, pBTrp2 (manufactured by Berliner Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238569) and the like can be mentioned.

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

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

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

The protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

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

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

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

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

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

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

<Protein fiber manufacturing method>
Protein fibers can be produced by known spinning methods. That is, for example, when producing a protein fiber containing a protein as a main component, first, a protein (for example, modified fibroin) produced according to the above-mentioned method is subjected to dimethyl sulfoxide (DMSO), N, N-dimethylformamide ( If necessary, it is added to a solvent such as DMF), formic acid, or hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator, and dissolved to prepare a dope solution. Then, using this dope solution, the desired protein fiber 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 protein fibers. The 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 moist heat drawing device 3, and a drying device 4.

A spinning method using the spinning device 10 will be described. First, the doping liquid 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8. On a lab scale, a cylinder may be filled with a dope solution and extruded from 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 protein is coagulated, and a fibrous coagulant is formed. Next, the fibrous solidified body is supplied into the hot water 12 in the stretching bath 21 and stretched. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14. Then, the stretched fibrous coagulant is supplied to the drying device 4 and dried in the yarn path 22, and the protein fiber 36 is obtained as the wound yarn body 5. 18a to 18g are thread guides.

The coagulation liquid 11 may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulating liquid 11 may contain water as appropriate. The temperature of the coagulating liquid 11 is preferably 0 to 30 ° C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the base 9, the extrusion speed is preferably 0.2 to 6.0 ml / hour, and 1.4 to 4.0 ml / hour per hole. More preferably, it is time. The distance through which the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the thread guide 18a to the thread guide 18b) may be as long as the solvent can be efficiently removed, for example, 200 to 500 mm. Is. The take-up 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 coagulating liquid 11. The coagulation liquid tank 20 may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage, if necessary.

As the stretching performed when obtaining the protein fibers, for example, in addition to the pre-stretching performed in the coagulation liquid tank 20 and the moist heat stretching performed in the stretching bath 21, dry heat stretching is also adopted.

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

Dry heat stretching can be performed using an electric tube 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 the pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.

Wet heat stretching and dry heat stretching may be performed individually, or they may be performed in multiple stages or in combination. That is, the first-stage stretching is performed by moist heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by moist heat stretching, the second stage stretching is performed by moist heat stretching, and the third stage stretching is performed by dry heat stretching. Wet heat stretching and dry heat stretching can be appropriately combined.

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

<Protein fiber>
The protein fiber according to the present embodiment may be a spun of the above-mentioned protein, and in this case, the above-mentioned protein is contained as a main component. Hereinafter, it is also simply referred to as "modified protein fiber", "modified fibroin fiber", "modified spider silk fibroin fiber", etc., depending on the protein used.

It is preferable that the protein fiber according to the present embodiment can be shrunk only by bringing it into contact with moisture to bring it into a wet state. Further, it is preferable that the protein fiber according to one embodiment can be contracted to a higher degree by contacting with water to bring it into a wet state and then drying it.

The protein fiber preferably has a shrinkage rate of 2% or more when wet. The shrinkage rate at the time of wetting is more preferably 4% or more, further preferably 6% or more, further preferably 8% or more, even more preferably 10% or more, and 15%. % Or more is particularly preferable, 20% or more is particularly more preferable, 25% or more is particularly more preferable, and 30% or more is most preferable. The upper limit of the shrinkage rate when wet is usually 80% or less. The shrinkage rate when wet is defined by the following formula II.
Shrinkage rate when wet = {1- (length of protein fiber moistened by contact with moisture / length of protein fiber before contact with moisture)} × 100 (%)… (Formula II)

The protein fiber preferably has a shrinkage rate of more than 7% when dried. The shrinkage rate during drying is more preferably 15% or more, further preferably 25% or more, further preferably 32% or more, even more preferably 40% or more, 48. % Or more is particularly preferable, 56% or more is particularly more preferable, 64% or more is particularly more preferable, and 72% or more is most preferable. The upper limit of the shrinkage rate during drying is usually 80% or less. The shrinkage rate during drying is defined by the following formula III.
Shrinkage rate during drying = {1- (length of protein fiber dried after being brought into contact with moisture / length of protein fiber before being brought into contact with moisture)} × 100 (%)… (formula III)

The protein fiber used for forming the raw material non-woven fabric may be a short fiber or a long fiber. In addition, protein fibers may be used alone or in combination with other fibers. That is, when producing a raw material non-woven fabric formed by using fibers containing at least a part of protein fibers, the fibers (also referred to as material yarns) include a single yarn composed of only protein fibers, protein fibers and other fibers. The composite yarns formed by combining the above may be used alone or in combination with each other. The single yarn and the composite yarn may be spun yarns in which short fibers are twisted, or filament yarns in which long fibers are twisted. Filament yarns are preferably used as the single yarns and the composite yarns. The other fibers refer to fibers containing no protein, and examples thereof include synthetic fibers such as nylon and polyester, recycled fibers such as cupra and rayon, and natural fibers such as cotton and hemp. When used in combination with other fibers, the content of protein fibers is preferably 5% by mass or more, more preferably 20% by mass or more, based on the total amount of fibers including protein fibers. It is more preferably 50% by mass or more. By setting the content of the protein fiber in the above range, the shrinkage rate in the shrinkage step of the raw material nonwoven fabric can be adjusted. Further, the composite yarn includes, for example, a blended yarn, a mixed fiber yarn, a covering yarn and the like.

<Manufacturing of raw material non-woven fabric>
The raw material non-woven fabric can be produced, for example, by using a fiber containing at least a part of protein fiber by a known method for producing a non-woven fabric. Specifically, for example, webs (including single-layer webs and laminated webs) are formed from fibers containing at least a part of protein fibers by a dry method, a wet method, an air-laid method, or the like, and then a chemical bond method (immersion) is performed. The raw material non-woven fabric can be obtained by bonding the fibers of the web by a method, a spray method, etc.) or a needle punching method.

Also, for example, the protein is added to a solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP), if necessary, with an inorganic salt as a dissolution accelerator. A raw material non-woven fabric can also be obtained as a nanofiber non-woven fabric by adding and dissolving to prepare a dope solution and then spinning the dope solution by an electrospinning method (electrostatic spinning method). The average diameter of nanofibers (average value of short fiber diameters) is usually 1000 nm or less, and may be 100 to 1000 nm, 200 to 900 nm, or 300 to 800 nm. The single fiber diameter of the nanofibers may vary between 100 and 1000 nm. Here, the single fiber diameter of the nanofiber means the diameter of the smallest circle surrounding the cross section perpendicular to the longitudinal direction of the short fiber.

The forming step may include adjusting the fiber density of the raw material nonwoven fabric (hereinafter, also referred to as “adjustment step”). The fiber density (basis weight) is a value defined by the weight per unit area of the non-woven fabric. By including the adjustment step, the fiber density of the high-density nonwoven fabric obtained by the method for producing the high-density nonwoven fabric in the present embodiment can be easily adjusted.

The fiber density of the raw material non-woven fabric can be adjusted, for example, by increasing or decreasing the amount of fibers constituting the web, or in the case of a laminated web, increasing or decreasing the number of layers.

(Shrinkage process)
The shrinkage step is a step of bringing the raw material non-woven fabric into contact with moisture (hereinafter, also referred to as “contact step”) to shrink the raw material non-woven fabric. Through such a shrinkage step, a high-density non-woven fabric can be obtained. In the shrinkage step, by bringing the raw material non-woven fabric into contact with water, the protein fibers can be shrunk (also referred to as water shrinkage) without relying on an external force, causing the entire non-woven fabric to shrink.

The contraction of protein fibers that is not due to external force is considered to occur, for example, for the following reasons. That is, one reason is considered to be due to the secondary or tertiary structure of the protein fiber, and another reason is that, for example, in a protein fiber having residual stress due to stretching in a manufacturing process or the like, water is a fiber. It is thought that it occurs because the residual stress is relaxed by invading the space or into the fiber. Therefore, it is considered that the shrinkage rate of the protein fiber in the shrinkage step can be arbitrarily controlled according to, for example, the magnitude of the draw ratio in the above-mentioned production process of the protein fiber. Further, the shrinkage rate of the protein fiber can be controlled by, for example, controlling the temperature of the water to be contacted, the contact time with the water, and the tensile force at the time of contact with the water in the shrinking step. Therefore, use a material yarn containing protein fibers whose shrinkage rate at the time of contact with moisture is adjusted by adjusting the draw ratio in the process of producing protein fibers, and the temperature of the moisture to be contacted in the shrinkage step and contact with moisture. The shrinkage rate of the raw material non-woven fabric due to contact with moisture can be arbitrarily adjusted by controlling the time, the tensile force at the time of contact with moisture, and the like. As a result, it is considered that a high-density non-woven fabric having a desired fiber density can be obtained.

Further, it is expected that the fiber density of the high-density non-woven fabric can be adjusted by appropriately selecting and using an appropriate yarn from material yarns having different shrinkage rates at the time of contact with moisture. Further, by limiting the amount of shrinkage of the raw material nonwoven fabric due to contact with moisture, the fiber density of the high-density nonwoven fabric can be adjusted regardless of the type of material yarn or the like. The method for limiting the amount of shrinkage of the raw material non-woven fabric due to contact with moisture is not particularly limited. Examples of the method for limiting the amount of shrinkage include a method in which the peripheral end portion of the raw material non-woven fabric is brought into contact with moisture in a fixed state and contracted. More specifically, the non-woven fabric obtained by contacting the non-woven fabric with the water and shrinking the maximum amount while the size is smaller than the original size of the non-woven fabric before the contact with the water and the peripheral end is free. In a state where the peripheral end of the raw material non-woven fabric is fixed on the entire circumference to a frame or the like having a size larger than the size by a predetermined size (a state in which shrinkage is allowed by the difference between the size of the frame and the size of the raw material non-woven fabric). The amount of shrinkage can be adjusted by bringing the raw material non-woven fabric into contact with water. By variously adjusting the size of such a frame, the fiber density of the high-density non-woven fabric can be arbitrarily adjusted.

As used herein, the term "moisture" means water in either a liquid or gaseous state. The method of bringing the moisture into contact with the raw material non-woven fabric in the contact step is also not particularly limited. For example, a method of immersing the raw material non-woven fabric in water, a method of spraying the raw material non-woven fabric in a state of normal temperature or heated steam, a method of exposing the raw material non-woven fabric in a high humidity environment filled with steam, and the like. Can be mentioned. Among these methods, the method of immersing the raw material non-woven fabric in water is preferable because the shrinkage time can be effectively shortened and the processing equipment can be simplified. As a method of immersing the raw material non-woven fabric in water, specifically, for example, a raw material non-woven fabric formed by using fibers containing protein fibers is put into a container containing water having a predetermined temperature. There is a method of contacting with water.

In the contact step, the temperature of water when the water is brought into contact with the raw material non-woven fabric is not particularly limited, but is preferably lower than the boiling point, for example. At such a temperature, handleability, workability in the shrinkage process, and the like are improved. The upper limit of the temperature of water is preferably 90 ° C. or lower, more preferably 80 ° C. or lower. The lower limit of the temperature of water is preferably 10 ° C. or higher, more preferably 40 ° C. or higher, and even more preferably 70 ° C. or higher. The temperature of the water in contact with the raw material non-woven fabric can be adjusted according to the fibers constituting the raw material non-woven fabric. Further, the temperature of the water may be constant while the water is brought into contact with the raw material non-woven fabric, or the temperature of the water may be changed so as to reach a predetermined temperature.

In the contact step, the time for bringing the moisture into contact with the raw material non-woven fabric is not particularly limited, and may be, for example, 1 minute or more. The time may be 10 minutes or more, 20 minutes or more, or 30 minutes or more. Further, the upper limit of the time is not particularly limited, but from the viewpoint of shortening the manufacturing process time and eliminating the risk of hydrolysis of protein fibers, for example, it may be 120 minutes or less, 90 minutes. It may be less than a minute or less than 60 minutes.

In addition to the contact step, the shrinkage step may further include contacting the raw material nonwoven fabric with moisture and then drying (hereinafter, also referred to as “drying step”).

The drying method in the drying step is not particularly limited, and for example, natural drying may be used, or forced drying may be performed using a drying facility. The drying temperature is not limited as long as it is lower than the temperature at which the proteins constituting the non-woven fabric are thermally damaged, but is generally in the range of 20 to 150 ° C. , The temperature is preferably in the range of 40 to 120 ° C, and more preferably the temperature is in the range of 60 to 100 ° C. Within such a temperature range, the non-woven fabric can be dried more quickly and efficiently without causing thermal damage to the protein or the like. The drying time is appropriately selected according to the drying temperature and the like, and for example, a time during which the influence on the quality and physical properties of the non-woven fabric due to overdrying of the protein fiber can be eliminated is adopted.

According to the above production method, since the fibers containing at least a part of the protein fibers are shrunk with water, the non-woven fabric having a higher fiber density than the conventional non-woven fabric obtained by simply forming using the fibers (high). Density non-woven fabric) can be produced. That is, according to the above-mentioned production method, it is possible to produce a high-density non-woven fabric having a fiber density exceeding the maximum value of the fiber density that can be realized by the non-woven fabric formed by the conventional technique using the fiber.

[High-density non-woven fabric]
As used herein, the term "high-density non-woven fabric" means a non-woven fabric having a high fiber density. For the high-density nonwoven fabric, as described above, the fiber density is the maximum value that can be produced by the conventional technique (the value of the production limit), in other words, the maximum value of the fiber density that can be achieved by the nonwoven fabric formed by the conventional method. Includes non-woven fabrics with fiber densities that exceed.

It fiber density of the high density non-woven fabric in accordance with the present embodiment, for example, be at 0.04 g / cm ² or more, preferably, 0.05 g / cm ² or more and 0.045 g / cm ² or more Is more preferable, and 0.055 g / cm ² or more is further preferable.

The high-density non-woven fabric according to the present embodiment preferably has a fiber density increase rate of 20% or more, more preferably 30% or more, further preferably 40% or more, and further preferably 50% or more. Is even more preferable, and 100% or more is even more preferable. The fiber density increase rate is a value defined by the following formula I or formula IV.
Fiber density increase rate = {(fiber density of high-density non-woven fabric / fiber density of raw non-woven fabric) -1} x 100 (%) ... (Formula I)
Fiber density increase rate = {(fiber density of high-density non-woven fabric after water shrinkage / fiber density of non-woven fabric before water shrinkage) -1} x 100 (%) ... (Equation IV)

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

[Manufacturing of modified spider silk fibroin (protein)]
(1) Construction of Modified Spider Fibroin Expression Vector Modified spider fibroin (PRT399) having the amino acid sequence shown in SEQ ID NO: 18, modified spider fibroin (PRT380) having the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13 A modified spider fibroin (PRT410) having the amino acid sequence shown in FIG. 15 and a modified spider fibroin (PRT799) having the amino acid sequence shown by SEQ ID NO: 15 were designed.

Nucleic acids encoding proteins having the designed amino acid sequence were synthesized. An NdeI site was added to the nucleic acid at the 5'end, and an EcoRI site was added downstream of the stop codon. These five types of nucleic acids were cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, respectively, and then recombinant into a protein expression vector pET-22b (+) to obtain an expression vector.

(2) Expression of modified spider silk fibroin Escherichia coli BLR (DE3) was transformed with 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 OD _{600 was 0.005.} The culture solution temperature was maintained at 30 ° C., _{and flask culture was carried out until the OD 600} reached 5, (about 15 hours) to obtain a seed culture solution.

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

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The culture solution temperature was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. The 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. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the desired modified spider silk fibroin. Twenty hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture solutions before and after the addition of IPTG, and the desired modification due to the appearance of a band of a size corresponding to the target modified spider fibroin depending on the addition of IPTG. The expression of spider silk fibroin was confirmed.

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

[Reference Example 1: Production of modified spider silk fibroin fiber (protein fiber) and evaluation of shrinkage]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and a freeze-dried powder of modified spider silk fibroin (PRT399, PRT380, PRT410 or PRT799) was added thereto at a concentration of 18% by mass. Alternatively, the mixture was added in an amount of 24% by mass and dissolved using a shaker for 3 hours. Then, insoluble matter and bubbles were removed to obtain a modified spider silk fibroin solution.

The obtained modified spider silk fibroin solution was used as a dope solution (spinning stock solution), and spun and stretched modified spider silk fibroin fibers were produced by dry-wet spinning using a spinning device according to the spinning device 10 shown in FIG. .. In the spinning device 10 shown in FIG. 6, the spinning device used was a second undrawn yarn manufacturing device (a second undrawn yarn manufacturing device) between the undrawn yarn manufacturing device 2 (first bath) and the moist heat drawing device 3 (third bath). It is equipped with a second bath). The conditions for dry-wet spinning are as follows.
Extrusion nozzle diameter: 0.2 mm
Coagulation bath temperature: 2 to 15 ° C
Total draw ratio: 1 to 4 times Drying temperature: 60 ° C

(Evaluation of shrinkage rate)
The shrinkage rate of the obtained modified spider silk fibroin fibers (Production Examples 1 to 19) was evaluated. That is, each modified spider silk fibroin fiber is brought into a wet state by contacting with water (contact step), and then dried (drying step), and a shrinkage step is carried out to bring the modified spider silk fibroin fiber into a wet state. , And the shrinkage rate of the modified spider silk fibroin fiber dried after being moistened was determined.

<Contact step>
From each wound of the modified spider silk fibroin fiber, a plurality of modified spider silk fibroin fibers having a length of 30 cm were cut out. The plurality of modified spider silk fibroin fibers were bundled to obtain a modified spider silk fibroin fiber bundle having a fineness of 150 denier. A 0.8 g lead weight was attached to each modified spider silk fibroin fiber bundle, and in that state, each modified spider silk fibroin fiber bundle was immersed in water at the temperatures shown in Tables 6 to 9 for 10 minutes. Then, the length of each modified spider silk fibroin fiber bundle was measured in water. The measurement was carried out with a 0.8 g lead weight attached to the modified spider silk fibroin fiber bundle in order to eliminate the crimp of the modified spider silk fibroin fiber bundle. Next, the shrinkage rate (shrinkage rate when wet) of the modified spider silk fibroin fiber in a wet state was calculated according to the following formula V. In the formula V, L0 indicates the length (30 cm) of the modified spider silk fibroin fiber bundle before being immersed in water, and Lw indicates the length of the modified spider silk fibroin fiber bundle that has been immersed in water and moistened.
Shrinkage rate when wet (%) = {1- (Lw / L0)} × 100 ... (Equation V)

<Drying step>
After the contact step, the modified spider silk fibroin fiber bundle was removed from the water. The removed modified spider silk fibroin fiber bundle was dried at room temperature for 2 hours with a 0.8 g lead weight attached. After drying, the length of each modified spider silk fibroin fiber bundle was measured. Next, the shrinkage rate (shrinkage rate at the time of drying) of the modified spider silk fibroin fiber that had been moistened and then dried was calculated according to the following formula VI. In the formula VI, L0 indicates the length (30 cm) of the modified spider silk fibroin fiber bundle before being immersed in water, and Lwd is the length of the modified spider silk fibroin fiber bundle that has been soaked in water to be moistened and then dried. Indicates the length.
Shrinkage rate during drying (%) = {1- (Lwd / L0)} x 100 (%) ... (Formula VI)

The results are shown in Tables 6-9. In Tables 6 to 9, "total draw ratio" indicates the total draw ratio in the spinning process.

From the above results, it can be seen that the modified spider silk fibroin fiber according to the present invention has a sufficiently high shrinkage rate. Therefore, it is recognized that the non-woven fabric formed from the modified spider silk fibroin fiber according to the present invention also has a sufficiently high shrinkage rate. The shrinkage ratio of the modified spider silk fibroin fiber and the non-woven fabric formed from the modified spider silk fibroin fiber according to the present invention is, for example, the draw ratio of the modified spider silk fibroin fiber, the temperature of the water to be contacted, the contact time with water, and the time of contact with water. It can also be understood that it can be controlled by controlling the tensile force.

[Example 1: Production and evaluation of highly shrinkable non-woven fabric]
(1) Production of raw material non-woven fabric Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and a freeze-dried powder of modified spider fibroin (PRT799) was added thereto at a concentration of 24 mass. It was added in% and dissolved for 3 hours using a shaker. Then, insoluble matter and bubbles were removed to obtain a modified spider silk fibroin solution.

The obtained modified spider silk fibroin solution was used as a dope solution (spinning stock solution), and dry-wet spinning was performed using a spinning apparatus according to the spinning apparatus 10 shown in FIG. 6, and modified spider yarn fibroin fibers (24 multifilaments). Got The conditions for dry-wet spinning are as follows.
Coagulant (methanol) temperature: 5-10 ° C
Stretching ratio: 5 times Drying temperature: 80 ° C

The obtained modified spider silk fibroin fiber (24 multifilaments) was cut to a predetermined length to shorten the fiber. Then, carding was performed with a known card machine to obtain a plurality of webs in which short fibers were entangled.

Next, one of the obtained webs was punched with a known needle punching machine to obtain a raw material non-woven fabric 1. The fiber density (weight) of this raw material non-woven fabric 1 was 3.5 g / m ² .

Further, three obtained webs are laminated in a state of being tilted at angles of 0 degree, 45 degree and 90 degree to obtain a laminated web, and then the laminated web is punched twice with a known needle punching machine to obtain a raw material. Nonwoven fabric 2 was obtained. The fiber density (weight) of the raw material non-woven fabric 2 was 5.3 g / m ² .

(2) Manufacture and Evaluation of High Shrink Nonwoven Fabrics Test pieces were cut out from the raw material nonwoven fabrics 1 and 2 to a size of 35 mm in length and 15 mm in width, and their weights were measured. Each of the test pieces was immersed in water at 40 ° C. for 10 minutes and then dried at room temperature for 12 hours to obtain two types of highly shrinkable nonwoven fabrics 1 and 2 having different fiber densities.

The vertical and horizontal lengths and weights of the obtained highly shrinkable nonwoven fabrics 1 and 2 were measured. Next, the shrinkage rate, fiber density, and fiber density increase rate of the high-shrinkable non-woven fabrics 1 and 2 were calculated, respectively (average value of n = 3). The results are shown in Table 10. The shrinkage rate and the fiber density increase rate were calculated according to the following formulas VII and I, respectively.
Shrinkage rate = {1- (length (horizontal) length of highly shrinkable non-woven fabric / length (horizontal) length of raw material non-woven fabric)} x 100 (%) ... (Formula VII)
Fiber density increase rate = {(fiber density of high-density non-woven fabric / fiber density of raw non-woven fabric) -1} x 100 (%) ... (Formula I)

According to the production method of the present invention, it is understood that a high-density non-woven fabric can be obtained only by a simple step of bringing the raw non-woven fabric (nonwoven fabric obtained according to a conventional method) into contact with moisture. In addition, the obtained high-density non-woven fabric shall achieve a fiber density exceeding the maximum value of the fiber density that can be achieved with the non-woven fabric (corresponding to the raw material non-woven fabric) obtained from the raw material fiber (modified spider silk fibroin fiber) according to a conventional method. It is also understood that can be done.

1 ... Extruder, 2 ... Undrawn yarn manufacturing equipment, 3 ... Wet and heat drawing equipment, 4 ... Drying equipment, 6 ... Dope liquid, 10 ... Spinning equipment, 20 ... Coagulation liquid tank, 21 ... Stretching bathtub, 36 ... Modified fibroin fiber ..

Claims (14)

A method for producing a high-density non-woven fabric, comprising a shrinkage step of bringing a raw non-woven fabric formed of fibers containing at least a part of protein fibers into contact with moisture to shrink the raw material non-woven fabric. The production method according to claim 1, wherein the fiber density increase rate defined by the following formula I is 20% or more.
Fiber density increase rate = {(fiber density of high-density non-woven fabric / fiber density of raw non-woven fabric) -1} x 100 (%) ... (Formula I) The production method according to claim 1 or 2, wherein the protein fiber has a shrinkage rate at wet time of 2% or more as defined by the following formula II.
Shrinkage rate when wet = {1- (length of protein fiber moistened by contact with moisture / length of protein fiber before contact with moisture)} × 100 (%)… (Formula II) The production method according to any one of claims 1 to 3, wherein the protein fiber has a shrinkage rate at drying of more than 7% as defined by the following formula III.
Shrinkage rate during drying = {1- (length of protein fiber dried after being brought into contact with moisture / length of protein fiber before being brought into contact with moisture)} × 100 (%)… (formula III) The production method according to any one of claims 1 to 4, wherein the temperature of the water is lower than the boiling point. Further provided with a forming process for forming the raw material non-woven fabric,
The production method according to any one of claims 1 to 5, wherein the forming step includes adjusting the fiber density of the raw material nonwoven fabric. The production method according to any one of claims 1 to 6, wherein the protein fiber is a fibroin fiber. The production method according to any one of claims 1 to 7, wherein the protein fiber is a modified spider silk fibroin fiber. A high-density non-woven fabric having fibers containing at least a part of protein fibers and the fibers being water-shrinked. A high-density non-woven fabric having fibers containing at least a part of protein fibers and being water-shrinked. The high-density nonwoven fabric according to claim 10, wherein the fiber density increase rate defined by the following formula IV is 20% or more.
Fiber density increase rate = {(fiber density of high-density non-woven fabric after water shrinkage / fiber density of non-woven fabric before water shrinkage) -1} x 100 (%) ... (Equation IV) It has fibers that contain at least a portion of protein fibers and
A high-density non-woven fabric in which the fiber density exceeds the maximum value of the fiber density that can be achieved with the non-woven fabric formed from the fibers. The high-density non-woven fabric according to any one of claims 9 to 12, wherein the protein fiber is a fibroin fiber. The high-density nonwoven fabric according to any one of claims 9 to 13, wherein the protein fiber is a modified spider silk fibroin fiber.

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