JP2020097796A - Artificial fibroin fiber - Google Patents

Abstract

To provide an artificial fibroin fiber that has a desired high contraction coefficient and can be produced safely.SOLUTION: An artificial fibroin fiber 36 of this invention includes modified fibroin and has a contraction coefficient larger than 7%, the contraction coefficient being defined by the following equation: contraction coefficient={1-(a length of the artificial fibroin fiber 36 subjected to contraction processing including being brought into contact with water at lower than the boiling point/a length of the artificial fibroin fiber 36 before subjected to the contraction processing)}×100(%). In the artificial fibroin fiber, a temperature of the water is 10-90°C, preferably 40-90°C, and more preferably 70-90°C and the modified fibroin preferably incudes modified spider silk fibroin. In the artificial fibroin fiber 36, the modified fibroin means fibroin in which an amino acid sequence of natural fibroin is modified, the modified fibroin is prepared by modifying nucleic acid coding the natural fibroin by a genetic engineering method or a chemical method, forming a vector log containing the modified sequence, and integrating the vector log into a host chromosome.SELECTED DRAWING: Figure 4

Description

The present invention relates to artificial fibroin fibers.

Generally, fibers used for clothing, bedding and the like are required to have high touch. Silk, which is one type of natural fibroin fibers, is known as a fiber that is sufficiently satisfying to the touch and has a high-grade feel.

In addition, the fibers used for clothing, bedding and the like are required to have softness and heat retention at the same time. Therefore, silk used for clothing, bedding, and the like may be subjected to shrinkage processing to increase its bulkiness, thereby imparting flexibility and heat retention.

On the other hand, for example, synthetic fibers such as polyester fibers, polyamide fibers, and acrylic fibers are generally used for clothes, bedding, etc. These synthetic fibers have a shrinkage ratio of 40% or more when they are brought into contact with boiling water. Has been realized (Patent Document 1).

JP, 2009-12103, A

However, the contraction rate of silk was only a few percent when it was contacted with water only.

Further, the shrinking method described in Patent Document 1 involves great danger because it handles high-temperature boiling water.

An object of the present invention is to provide an artificial fibroin fiber which has a sufficiently high shrinkage factor and can be manufactured safely.

The present invention relates to the following inventions, for example.
[1]
Man-made fibroin fiber containing modified fibroin and having a shrinkage ratio defined by the following formula of more than 7%.
Shrinkage rate={1-(length of artificial fibroin fiber that has been subjected to shrinkage treatment including contact with water having a boiling point lower than that/length of artificial fibroin fiber before the above-mentioned shrinkage treatment)}×100 (%)
[2]
The artificial fibroin fiber according to [1], wherein the modified fibroin is a modified spider silk fibroin.
[3]
The artificial fibroin fiber according to [1] or [2], wherein the temperature of the water is 10 to 90°C.
[4]
The artificial fibroin fiber according to any one of [1] to [3], wherein the shrinking process includes drying after contact with the water.

According to the present invention, it is possible to provide an artificial fibroin fiber which has a sufficiently high shrinkage factor and can be manufactured safely.

It is a schematic diagram which shows the domain sequence of a modified fibroin. It is a figure which shows the distribution of the value of z/w (%) of the fibroin of natural origin. It is a figure which shows the distribution of the value of x/y (%) of fibroin derived from nature. It is an explanatory view showing roughly an example of the spinning device for manufacturing artificial fibroin fiber. It is explanatory drawing which shows roughly an example of the apparatus for shrink-processing an artificial fibroin fiber. It is explanatory drawing which shows roughly an example of the apparatus for shrink-processing an artificial fibroin fiber.

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

[Artificial fibroin fiber]
The artificial fibroin fiber according to the present invention contains modified fibroin and is capable of high shrinkage simply by contacting with water (water, steam, etc.). High shrinkage means that the shrinkage rate defined by the following formula exceeds 7%.
Shrinkage rate={1-(length of artificial fibroin fiber that has been subjected to shrinkage processing including contact with water having a temperature lower than boiling point/length of artificial fibroin fiber before shrinkage processing)}×100 (%)

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

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

The “modified fibroin” may be the one that directly uses the amino acid sequence of naturally occurring fibroin as long as it has the amino acid sequence specified in the present invention, and depends on the amino acid sequence of naturally occurring fibroin. The amino acid sequence may be modified (for example, the amino acid sequence is modified by modifying the gene sequence of cloned naturally-occurring fibroin), or artificially designed and independent of naturally-occurring fibroin. It may be synthetic (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

As used herein, the term “domain sequence” refers to a crystalline region (typically corresponding to the (A) _n motif of an amino acid sequence) and an amorphous region (typically, a REP of an amino acid sequence) peculiar to fibroin. Corresponding to the amino acid sequence represented by formula 1: [(A) _n motif-REP] _m . Here, the (A) _n motif represents an amino acid sequence composed of 4 to 20 amino acid residues, and the number of alanine residues is 83% or more based on the total number of amino acid residues in the (A) _n motif. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 8 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.

In the (A) _n motif, the number of alanine residues to the total number of amino acid residues in the (A) _n motif may be 83% or more, preferably 86% or more, and preferably 90% or more. It is more preferably 95% or more, still more preferably 100% (meaning that it is composed of only alanine residues). It is preferable that at least seven of the (A) _n motifs present in the domain sequence are composed of only alanine residues. The expression (A) _n motif _means that (A) _n (A represents an alanine residue, n is an integer of 2 to 20, preferably 4 to 20, and more preferably 4 to 4). It means having an amino acid sequence represented by 16).

The modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to the one obtained by substituting, deleting, inserting and/or adding one or more amino acid residues with respect to the gene sequence of the cloned naturally-derived fibroin. It can be obtained by modifying. Amino acid residue substitutions, deletions, insertions and/or additions can be made by methods well known to those skilled in the art such as partial directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983) and the like.

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

Examples of the fibroin produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yam tai cya cya pyramid, Anteraea pya rya pyramid, Anteraea peri, rye, Pomegranate (Anteraea periy), Anteraea peryny (Etera peri), Pomegranate (Anteraea pernii), ), silk proteins produced by silkworms such as Anthera pear insects (Antheraea assasa), sperm silkworms produced by silkworms such as Anthera apia nets (Antheraea nets). Silk protein is mentioned.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), AAA278840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include spiders belonging to the genus Araneus (genus Araneus) such as Onigumo, Niwaonimogu, Akanonigumo, Aonigumo and Maeoniguimo, spiders of the genus Araneus, spiders of the genus Nectar, e. Spiders belonging to the genus Proton, spiders belonging to the genus Pronus, spiders belonging to the genus Cyrtarachne, such as Asterinofundamas and Otorinofundamas, and the thorn spiders such as Spiders Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius such as Mameitai Seki spider and Mutsugai spider, and belonging to the genus Argiopsis such as Argiogiope, Argiope spp. Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas such as the spider Spider, spiders belonging to the genus Cytophora, such as the spider Spider, the brown spider, and the spider Spider (genus Cytophora). Spider silk protein produced by spiders belonging to the genus Cyclopsa such as spiders belonging to the category ), spider silk proteins such as the spider silk protein belonging to the genus Cyclosa, and spider silk proteins produced by the spider belonging to the genus Chorizopes, such as the spider silk spider. Spiders belonging to the genus Tetragnatha, such as the herring-backed spider, sword-tailed duck spider and urocore-ringed spider, etc. Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira, such as the black spider, spiders belonging to the genus Dyschiriognatha, such as the dwarf spider, such as the black raccoon spider, the black widow spider, the black-brown spider, and the spider black spider. Spiders produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus (genus Latrodetectus) and spiders belonging to the genus Euprosthenops (genus Euprosthenops). Examples include pider silk protein. Examples of the spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of fibroin produced by spiders include, for example, fibroin-3 (adf-3) [from Araneus diadematus] (GenBank Accession No. AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-. 4(adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenACbank accession number 4 amino acid sequence ANC50) U37520 (base sequence)), major amplifier spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein splatinbal2a2c [Negative]. (Amino acid sequence), AF441245 (base sequence), major amplifier spidroin 1 [derived from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampulth luteurospinuroin 2 (base sequence). Accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampulate silk NP [2]. (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ample spidroin-like protein [Nephilengys cruenta] (GenBank Accession No. ABR). 37278.1 (amino acid sequence) and the like.

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

The modified fibroin according to the present embodiment may be modified silk fibroin (modified amino acid sequence of silk protein produced by silkworm), and modified spider silk fibroin (amino acid sequence of spider silk protein produced by spiders). Modified).

As a specific example of the modified fibroin according to the present embodiment, modified fibroin having a reduced content of glycine residues (modified fibroin according to the first embodiment), the content of (A) _n motif was reduced. Modified fibroin (modified fibroin according to the second embodiment), modified fibroin having a reduced glycine residue content and (A) _n motif content (modified fibroin according to the third embodiment) can be mentioned. ..

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

In the modified fibroin according to the first embodiment, its domain sequence is at least selected from GGX and GPGXX in REP (where X represents an amino acid residue other than glycine), as compared with naturally occurring fibroin. One motif sequence may have an amino acid sequence corresponding to at least one or more glycine residues in the motif sequence being replaced with another amino acid residue.

In the modified fibroin according to the first embodiment, the proportion of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more with respect to the entire motif sequence.

The modified fibroin according to the first embodiment includes a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and the (A) _n motif located closest to the C-terminal side from the domain sequence. To z, the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the above to the C-terminal of the domain sequence is defined as z. When the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located closest to the C terminus to the C terminus of the domain sequence in the above domain sequence is w, z/w is It may have an amino acid sequence of 30% or more, 40% or more, 50% or more, or 50.9% or more.

The modified fibroin according to the first embodiment is obtained by replacing one glycine residue of the GGX motif with another amino acid residue. It is preferable that the content ratio of the amino acid sequence consisting of XGX is increased. In the modified fibroin according to the first embodiment, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, and more preferably 10% or less. It is more preferably 6% or less, still more preferably 4% or less, still more preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method of 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, the amino acid sequence consisting of XGX is extracted from all the REPs contained in the sequence excluding the sequence from the (A) _n motif located closest to the C terminus to the C terminus 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 have been extracted (no duplication), z is 50×3=150. Further, for example, when there is an X (center X) contained in two XGXs, as in the case of an amino acid sequence consisting of XGXGX, calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used. is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located closest to the C terminus to the C terminus 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 at the most C-terminal side). Then, z/w (%) can be calculated by dividing z by w.

Here, z/w in naturally occurring fibroin will be described. First, as described above, when confirmed by a method exemplifying fibroin having amino acid sequence information registered in NCBI GenBank, 663 kinds of fibroins (of which 415 kinds of fibroins derived from arachnids) were extracted. Of all the extracted fibroins, the naturally-occurring origin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and the content of the amino acid sequence consisting of GGX in fibroin is 6% or less. Z/w was calculated from the amino acid sequence of fibroin of No. 1 by the above-mentioned calculation method. The result is shown in FIG. The horizontal axis of FIG. 2 represents z/w (%), and the vertical axis represents frequency. As is clear from FIG. 2, z/w in naturally-derived fibroin is less than 50.9% (50.86% at the highest).

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

Modified fibroin according to the first embodiment, for example, so as to encode another amino acid residue by substituting at least a part of the nucleotide sequence encoding a glycine residue from the cloned gene sequence of naturally-occurring fibroin. It can be obtained by modification. 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 the glycine residue may be substituted so that z/w is 50.9% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence satisfying the above-mentioned aspect from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , The amino acid sequence corresponding to the insertion and/or addition may be modified.

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

As a more specific example of the modified fibroin according to the first embodiment, (1-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or the (1-ii) sequence. A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 can be mentioned.

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

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

The modified fibroin of (1-i) may consist of the amino acid sequence shown by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin of (1-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (1-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 (1-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is contained in REP (XGX( Where X is an amino acid residue other than glycine), and z is the total number of amino acid residues of the amino acid sequence consisting of) and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.

The modified fibroin described above may include a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, etc. of modified fibroin.

As the tag sequence, for example, an affinity tag utilizing specific affinity (binding property, affinity) with another molecule can be mentioned. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are lined up and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography is performed. Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence containing His tag).

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

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

Further, a tag sequence that can be cleaved with 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 is cleaved can be recovered.

As a more specific example of the modified fibroin containing a tag sequence, (1-iii) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or the amino acid sequence shown by SEQ ID NO: 13 or (1-iv) SEQ ID NO: 8 , SEQ ID NO: 9, SEQ ID NO: 11 or a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13.

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

The modified fibroin of (1-iii) may consist of the amino acid sequence shown by SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

The modified fibroin of (1-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (1-iv) 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 (1-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and is contained in REP (XGX( Where X is an amino acid residue other than glycine), and z is the total number of amino acid residues of the amino acid sequence consisting of) and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.

The modified fibroin described above 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 secretion signal can be appropriately set depending on the type of host.

The modified fibroin according to the second embodiment has an amino acid sequence whose domain sequence has a reduced content of the (A) _n motif as compared to naturally-occurring fibroin. It can be said that the domain sequence of the modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) _n motifs as compared with the naturally occurring fibroin.

The modified fibroin according to the second embodiment may have an amino acid sequence corresponding to the (A) _n motif deleted from natural fibroin by 10 to 40%.

In the modified fibroin according to the second embodiment, the domain sequence has one to three (A) _n motifs at least from the N-terminal side toward the C-terminal side as compared with naturally-occurring fibroin ( A) It may have an amino acid sequence corresponding to the deletion of the _n motif.

In the modified fibroin according to the second embodiment, the domain sequence of the modified fibroin is at least two consecutive (A) _n motifs deleted from the N-terminal side toward the C-terminal side as compared with naturally-occurring fibroin, and It may have an amino acid sequence corresponding to the deletion of one (A) _n motif repeated in this order.

The modified fibroin according to the second embodiment has a domain sequence having an amino acid sequence corresponding to at least every two (A) _n motifs deleted from the N-terminal side toward the C-terminal side. It may be.

The modified fibroin according to the second embodiment includes a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and has two adjacent [[( A) The number of amino acid residues of REP in the _n motif-REP] unit is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues of the other REP is Let x be the maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) _n motif-REP] units of 1.8 to 11.3, and the total number of amino acid residues of the domain sequence. When y is defined, x/y may have an amino acid sequence of 20% or more, 30% or more, 40% or more, or 50% or more.

The method of calculating x/y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from modified fibroin. The domain sequence is (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n from the N-terminal side (left side). 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 n} motif sequence.

Two adjacent [(A) _n motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, an unselected [(A) _n motif-REP] unit may exist. In FIG. 1, pattern 1 (comparison of first REP and second REP and comparison of third REP and fourth REP), pattern 2 (comparison of first REP and second REP, and Pattern 4 (comparison of fourth REP and fifth REP), pattern 3 (comparison of second REP and third REP, and comparison of fourth REP and fifth REP), pattern 4 (compared with first REP) (Comparison of the second REP). There are other selection methods besides this.

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 determining the ratio of the number of amino acid residues of the other when the number of amino acid residues having a smaller number is 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, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is 1, the fifth REP The ratio of the number of amino acid residues is 30/20=1.5.

In FIG. 1, when the one having the smaller number of amino acid residues is set to 1, the ratio of the number of the other amino acid residues is 1.8 to 11.3, and the set of [(A) _n motif-REP] units is It is shown by a solid line. Hereinafter, such a ratio is referred to as a knurled ratio. The set of [(A) _n motif-REP] units in which the ratio of the number of other amino acid residues is less than 1.8 or more than 11.3 is indicated by a broken line when the number of amino acid residues is 1 Indicated.

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

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

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

When the modified fibroin according to the second embodiment is a modified fibroin in which at least 7 of (A) _n motifs present in plural in the domain sequence are composed of only alanine residues, x/y is 46.4%. It is preferably at least 50%, more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, even more preferably at least 70%, It is particularly preferably 80% or more. The upper limit of x/y is not particularly limited and may be 100% or less.

Here, x/y in naturally occurring fibroin will be described. First, as described above, when confirmed by a method exemplifying fibroin having amino acid sequence information registered in NCBI GenBank, 663 kinds of fibroins (of which 415 kinds of fibroins derived from arachnids) were extracted. Of all the extracted fibroins, x/y was calculated from the amino acid sequence of naturally-derived 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 serrated ratio is 1:1.9 to 4.1 are shown in FIG.

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

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

As a more specific example of the modified fibroin according to the second embodiment, (2-i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2-ii) sequence. A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 can be mentioned.

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

The x/y value of the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally occurring fibroin) at the Giza ratio of 1:1.8 to 11.3 is 15.0%. The values of x/y in the amino acid sequence shown by SEQ ID NO:2 and the amino acid sequence shown by SEQ ID NO:4 are both 93.4%. The value of x/y in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%. The value of x/y in the amino acid sequence represented by SEQ ID NO: 12 is 89.8%. The values of z/w in the amino acid sequences shown in SEQ ID NOs: 1, 2, 4, 10 and 12 were 46.8%, 56.2%, 70.1%, 66.1% and 80.4%, respectively. is there.

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

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by 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 shown by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and from the N-terminal side to the C-terminal side. Toward, the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is 1, the other 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) It is preferable that x/y is 64.2% or more, where x is the maximum value of the total sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.

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

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

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

The modified fibroin of (2-iii) may consist of the amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The modified fibroin (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by 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 represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and from the N-terminal side to the C-terminal side. Toward, the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is 1, the other X is the maximum value of the sum of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units in which the ratio of the amino acid residue numbers of the REPs 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 modified fibroin described above 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 secretion signal can be appropriately set depending on the type of host.

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

As a more specific example of the modified fibroin according to the third embodiment, (3-i) SEQ ID NO: 4, amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12, (3-ii) SEQ ID NO: 4, SEQ ID NO: 10 or modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12. Specific embodiments of the modified fibroin containing the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.

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

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

The regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of the host. As a promoter, an inducible promoter that functions in a host cell and is capable of inducing expression of modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducer (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, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, or the like. As the expression vector, one that can autonomously replicate in a host cell or can be integrated into the host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.

As a host, any of prokaryote and eukaryote such as yeast, filamentous fungus, insect cell, animal cell and plant cell can be preferably used.

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

When a prokaryote is used as a host, examples of vectors for introducing a nucleic acid encoding modified fibroin include pBTrp2 (manufactured by Boehringer 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.

Examples of eukaryotic hosts include yeast and filamentous fungi (mold and the like). Examples of yeasts include yeasts belonging to the genera Saccharomyces, Pichia, Schizosaccharomyces, and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

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

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

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

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

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

Cultivation of prokaryotic organisms such as Escherichia coli or eukaryotic organisms such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration agitation culture. The culture temperature is, for example, 15 to 40°C. Cultivation 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 inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like.

During the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium, 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, if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used, and when culturing a microorganism transformed with the expression vector using the trp promoter, indole acrylic is used. You may add an acid etc. to a culture medium.

The expressed modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in cells, after the culture is completed, the host cells are recovered by centrifugation, suspended in an aqueous buffer solution, and then ultrasonically disrupted, French press, Menton. A cell-free extract is obtained by crushing host cells with a Gaurin homogenizer, Dynomill and the like. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying modified fibroin, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent is used. By anion-exchange chromatography using a resin such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), or a resin such as S-Sepharose FF (manufactured by Pharmacia). Electrophoresis such as cation exchange chromatography, butyl sepharose and phenyl sepharose, hydrophobic chromatography using resins, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. A purified sample can be obtained by using methods such as the method alone or in combination.

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

<Manufacturing method of artificial fibroin fiber>
The artificial fibroin fiber according to the present embodiment can be manufactured by a known spinning method. That is, for example, when manufacturing an artificial fibroin fiber containing modified fibroin as a main component, first, modified fibroin produced according to the above-mentioned method is treated with dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), Alternatively, a dope solution is prepared by adding it to a solvent such as hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator and dissolving it. Then, the dope solution can be spun by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning to obtain the target artificial fibroin fiber. As a preferable spinning method, wet spinning or dry wet spinning can be mentioned.

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

A spinning method using the spinning device 10 will be described. First, the dope liquid 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8. In the laboratory scale, the dope solution may be filled in a cylinder and pushed out from a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied into the coagulating liquid 11 in the coagulating liquid tank 20 through the air gap 19, the solvent is removed, and the modified fibroin is coagulated to form a fibrous coagulated body. Next, the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14. After that, the stretched fibrous solidified body is supplied to the drying device 4 and dried in the yarn path 22 to obtain the artificial fibroin fiber as the wound body 5. 18a-18g are yarn 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 coagulation liquid 11 may appropriately contain water. The temperature of the coagulating liquid 11 is preferably 0 to 30°C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the mouthpiece 9, the extrusion rate is preferably 0.2 to 6.0 ml/hour per hole, 1.4 to 4.0 ml/hour. More preferably it is time. The distance that the coagulated protein passes through the coagulation liquid 11 (substantially the distance from the thread guide 18a to the thread guide 18b) may be such that desolvation can be efficiently performed, and 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 coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or in a specific stage as needed.

The stretching performed when obtaining the artificial fibroin fiber includes, for example, dry stretching, as well as the above-described pre-stretching performed in the coagulating liquid tank 20 and wet-heat stretching performed in the stretching bath 21.

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

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

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

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 that of the undrawn yarn (or pre-drawn yarn). Any of the above, 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less , 12 times or less, 11 times or less, and 10 times or less.

<Artificial fibroin fiber>
The artificial fibroin fiber according to the present embodiment is obtained by spinning the modified fibroin described above, and contains the modified fibroin described above as a main component.

The artificial fibroin fiber according to the present embodiment preferably has a shrinkage rate defined by the following formula of more than 7%. As a result, it is possible to shrink at a higher shrinkage rate than conventionally known protein fibers, and at a higher shrinkage rate under milder conditions than conventionally known synthetic fibers.
Shrinkage rate={1-(length of artificial fibroin fiber that has been subjected to shrinkage processing including contact with water having a temperature lower than boiling point/length of artificial fibroin fiber before shrinkage processing)}×100 (%)

The shrinkage rate of the artificial fibroin fiber according to the present embodiment is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, further preferably 40% or more. More preferably, it is still more preferably 48% or more, still more preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more. The upper limit of shrinkage is usually 80% or less.

<Shrink processing>
The shrinking process includes contacting the artificial fibroin fiber with water having a temperature lower than the boiling point (hereinafter, also referred to as “contact step”). The shrinking process may further include drying the artificial fibroin fiber after contact with water (after the contact step) (hereinafter, also referred to as “drying step”).

The temperature of the water contacted with the artificial fibroin fiber in the contacting step may be below the boiling point. As a result, the handleability and workability of shrink processing are improved. From the viewpoint of sufficiently shortening the shrinkage time, the lower limit of the water temperature is preferably 10°C or higher, more preferably 40°C or higher, and further preferably 70°C or higher. .. The upper limit of the water temperature is preferably 90°C or lower.

The method of contacting the artificial fibroin fiber with water in the contacting step is not particularly limited. As the method, for example, a method of immersing the artificial fibroin fiber in water, a method of spraying water to the artificial fibroin fiber at room temperature or in a heated state such as steam, and high humidity filled with artificial fibroin fiber steam. Examples include the method of exposure to the environment. Among these methods, in the contacting step, the method of immersing the artificial fibroin fiber in water is preferable because the contraction time can be effectively shortened and the processing equipment can be simplified.

When the artificial fibroin fiber is brought into contact with water in a relaxed state in the contacting step, the artificial fibroin fiber may not only be contracted but may be wrinkled. In order to prevent the occurrence of such crimps, for example, the contact step is performed in a state where the artificial fibroin fiber is not relaxed, such as contacting the artificial fibroin fiber with water below the boiling point while tensioning (pulling) the fiber axis direction. May be.

The drying step is a step of drying the artificial fibroin fiber that has undergone the contacting step. Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known contact equipment or non-contact equipment can be used. Further, the drying temperature is not limited, for example, as long as it is a temperature lower than the temperature at which the protein contained in the artificial fibroin fiber is decomposed or the artificial fibroin fiber is thermally damaged, but in general, The temperature is in the range of 20 to 150°C, preferably the temperature in the range of 50 to 100°C. When the temperature is in this range, the artificial fibroin fiber is dried more quickly and efficiently without causing thermal damage to the artificial fibroin fiber or decomposition of the protein contained in the artificial fibroin fiber. The drying time is appropriately set according to the drying temperature and the like, and for example, the time and the like in which the influence on the quality and physical properties of the artificial fibroin fiber due to overdrying can be eliminated as much as possible are adopted.

[High shrinkage artificial fibron fiber]
Since the artificial fibroin fiber according to the present invention is capable of high shrinkage, for example, the highly shrinkable artificial fibroin fiber can be obtained through the above-described shrinking process (contact step and, if necessary, drying step).

The high shrinkage artificial fibroin fiber according to the present embodiment has a shrinkage rate as defined above of more than 7%. The shrinkage ratio of the highly shrinkable artificial fibroin fiber is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, further preferably 40% or more, It is even more preferably 48% or more, particularly preferably 56% or more, particularly preferably 64% or more, most preferably 72% or more. The upper limit of shrinkage is usually 80% or less.

FIG. 5: is explanatory drawing which shows schematically an example of the apparatus for shrink-processing an artificial fibroin fiber. An apparatus 40 shown in FIG. 5 includes a feed roller 42 that sends out artificial fibroin fibers, a winder 44 that winds up the highly-shrinkable artificial fibroin fibers 38, a water bath 46 that performs a contact step, and a dryer 48 that performs a drying step. , And are configured.

More specifically, the wound product of the artificial fibroin fiber 36 can be mounted on the feed roller 42, and the artificial fibroin fiber 36 is continuously fed from the wound product of the artificial fibroin fiber 36 by rotation of an electric motor or the like (not shown). It can be sent out automatically and automatically. The winder 44 is capable of continuously and automatically winding the high shrinkage artificial fibroin fiber 38 produced through the contact step and the drying step after being delivered from the feed roller 42, by the rotation of an electric motor (not shown). ing. Here, the feeding speed of the artificial fibroin fiber 36 by the feed roller 42 and the winding speed of the high shrinkage artificial fibroin fiber 38 by the winder 44 can be controlled independently of each other.

The water bath 46 and the dryer 48 are arranged side by side between the feed roller 42 and the winder 44 on the upstream side and the downstream side in the feeding direction of the artificial fibroin fiber 36, respectively. The device 40 shown in FIG. 5 has relay rollers 50 and 52 that relay the artificial fibroin fibers 36 running from the feed roller 42 toward the winder 44.

The water bath 46 has a heater 54, and hot water 47 heated by the heater 54 is contained in the water bath 46. A tension roller 56 is installed in the water bath 46 in a state of being immersed in the hot water 47. As a result, the artificial fibroin fiber 36 delivered from the feed roller 42 travels toward the winder 44 side while being immersed in the hot water 47 in the water bath 46 while being wound around the tension roller 56. Has become. In addition, the immersion time of the artificial fibroin fiber 36 in the hot water 47 is appropriately controlled according to the traveling speed of the artificial fibroin fiber 36.

The dryer 48 has a pair of hot rollers 58. The pair of hot rollers 58 can be wound around the artificial fibroin fiber 36 that moves away from the water bath 46 toward the winder 44 side. As a result, the artificial fibroin fiber 36 immersed in the hot water 47 in the water bath 46 is heated by the pair of hot rollers 58 in the dryer 48, dried, and then further delivered to the winder 44. It has become.

When manufacturing the high shrinkage artificial fibroin fiber 38 using the device 40 having such a structure, first, for example, winding of the artificial fibroin fiber 36 spun using the spinning device 10 shown in FIG. The scroll is attached to the feed roller 42. Next, the artificial fibroin fiber 36 is continuously sent out from the feed roller 42 and immersed in the hot water 47 in the water bath 46. At this time, for example, the winding speed of the winder 44 is set lower than the feeding speed of the feed roller 42. As a result, the artificial fibroin fiber 36 contracts due to contact with the hot water 47 in a state in which the artificial fibroin fiber 36 is strained so as not to relax between the feed roller 42 and the winder 44, and thus the occurrence of shrinkage can be prevented.

Next, the artificial fibroin fiber 36 contracted in the hot water 47 in the water bath 46 is heated by the pair of hot rollers 58 of the dryer 48. As a result, the contracted artificial fibroin fiber 36 is dried to form the highly contracted artificial fibroin fiber 38. At this time, by controlling the ratio of the feeding speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 36 can be further shrunk and the length can be not changed. Then, the obtained high shrinkage artificial fibroin fiber 38 is wound by a winder 44 to obtain a wound product of the high shrinkage artificial fibroin fiber 38.

Instead of the pair of hot rollers 58, the artificial fibroin fiber 36 may be dried using a dry heat plate 64 as shown in FIG. Also in this case, the relative speeds of the feed speed of the feed roller 42 and the winding speed of the winder 44 are adjusted in the same manner as in the case of using the pair of hot rollers 58 as the drying equipment, so that the artificial fibroin fiber is obtained. It is possible for 36 to be further shrunk and the length not to change. Here, the drying means is composed of the dry heat plate 64.

As described above, by using the apparatus 40, the desired highly-shrinkable artificial fibroin fiber 38 can be produced automatically and continuously and extremely easily.

FIG. 6 is an explanatory view schematically showing another example of the apparatus for shrink-processing the artificial fibroin fiber. FIG. 6( a) shows a processing apparatus provided in the apparatus for performing the contact step, and FIG. 6( b) shows a drying apparatus provided in the apparatus for performing the drying step. The apparatus shown in FIG. 6 has a processing device 60 for performing a contact step with respect to the artificial fibroin fiber 36, and a drying device 62 for drying the artificial fibroin fiber 36 subjected to the contact step with the processing device 60. Are independent of each other.

More specifically, in the processing device 60 shown in FIG. 6A, the dryer 48 is omitted from the device 40 shown in FIG. 5, and the feed roller 42, the water bath 46, and the winder 44 are connected to the artificial fibroin. The fiber 36 has a structure in which the fibers 36 are arranged in order from the upstream side to the downstream side in the traveling direction. In the processing device 60, the artificial fibroin fiber 36 sent out from the feed roller 42 is contracted by being immersed in the hot water 47 in the water bath 46 before being wound by the winder 44. .. The artificial fibroin fiber 36 contracted in the hot water 47 is wound by the winder 44.

The drying device 62 shown in FIG. 6B has a feed roller 42, a winder 44, and a dry heat plate 64. The dry heat plate 64 is arranged between the feed roller 42 and the winder 44 so that the dry heat surface 66 is in contact with the artificial fibroin fiber 36 and extends along the traveling direction. In the drying device 62, as described above, for example, by controlling the ratio between the feeding speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 36 can be further shrunk, and the length of the artificial fibroin fiber 36 can be reduced. It is possible not to change the height.

When an apparatus having such a structure is used, for example, first, the artificial fibroin fiber 36 is contracted by the processing device 60, and then the artificial fibroin fiber 36 is dried by the drying device 62 to obtain the object. The high shrinkage artificial fibroin fiber 38 can be manufactured.

The feed roller 42 and the winder 44 may be omitted from the processing device 60 shown in FIG. 6A, and the processing device may be configured by only the water bath 46. When an apparatus having such a processing apparatus is used, for example, the high shrinkage artificial fibroin fiber is manufactured by a so-called batch method.

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

[(1) Production of modified fibroin (artificial fibroin)]
(Synthesis of nucleic acid encoding modified fibroin and construction of expression vector)
Modified fibroin having the amino acid sequence represented by SEQ ID NO: 7 (PRT399), modified fibroin having the amino acid sequence represented by SEQ ID NO: 8 (PRT380), modified fibroin having the amino acid sequence represented by SEQ ID NO: 9 (PRT410), SEQ ID NO: A modified fibroin (PRT799) having the amino acid sequence shown by 13 was designed.

The amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally-occurring fibroin obtained by deleting every two (A) _n motifs from the N-terminal side toward the C-terminal side. Furthermore, the amino acid sequence in which one [(A) _n motif-REP] is inserted in front of the C-terminal sequence has the amino acid sequence represented by SEQ ID NO: 5 (including His tag) added at the N-terminal. ..

The amino acid sequence shown by SEQ ID NO:8 is shown at the N-terminal by SEQ ID NO:5 with respect to the amino acid sequence obtained by substituting GQX for all GGX in REP of the amino acid sequence shown by SEQ ID NO:1 corresponding to naturally-occurring fibroin. The amino acid sequence (including His tag) is added.

The amino acid sequence represented by SEQ ID NO: 9 is the amino acid sequence represented by SEQ ID NO: 5 at the N-terminal (amino acid sequence represented by SEQ ID NO: 5 at the N-terminal of the amino acid sequence obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 2 with GQX). (Including) is added.

The amino acid sequence represented by SEQ ID NO: 13 is a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which the His tag is added to the C-terminal of the sequence repeated 4 times, and the amino acid sequence shown in SEQ ID NO: 5 (including the His tag) is added to the N-terminal.

Nucleic acids encoding the designed four types of modified fibroin were respectively synthesized. An NdeI site was added to the 5'end and an EcoRI site was added downstream of the stop codon to the nucleic acid. These four kinds of nucleic acids were cloned into a cloning vector (pUC118). Then, the same nucleic acid was digested with restriction enzymes NdeI and EcoRI and cut out, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

(Expression of modified fibroin)
Escherichia coli BLR(DE3) was transformed with the obtained pET-22b(+) expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium containing ampicillin (Table 1) so that the OD ₆₀₀ was 0.005. The temperature of the culture solution was maintained at 30° C., and flask culture was performed until the OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter to which 500 ml of the production medium (Table 2 below) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was kept at 37° C., and the culture was performed at a constant pH of 6.9. Culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce the expression of the target modified fibroin. 20 hours after the addition of IPTG, the culture solution was centrifuged to collect the bacterial cells. SDS-PAGE is performed using the cells prepared from the culture medium before and after the addition of IPTG, and the appearance of a band having a size corresponding to the target modified fibroin depending on the IPTG addition causes the formation of the target modified fibroin. The expression was confirmed.

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

The degree of purification of the target modified fibroin in the obtained lyophilized powder was confirmed by image analysis of the result of polyacrylamide gel electrophoresis of the powder using Totallab (nonlinear dynamics ltd.). As a result, the purity of any modified fibroin was about 85%.

[(2) Manufacture of artificial fibroin fiber]
(Preparation of dope solution)
Dimethylsulfoxide (DMSO) in which LiCl was dissolved so as to have a concentration of 4.0 mass% was prepared as a solvent, and lyophilized powder of modified fibroin was added thereto at a concentration of 18 mass% or 24 mass% (Table 3), and dissolved for 3 hours using a shaker. Then, insoluble matter and bubbles were removed to obtain a modified fibroin solution.

(spinning)
The modified fibroin solution thus obtained was used as a dope solution (spinning stock solution), and by dry-wet spinning using a spinning device similar to the spinning device 10 shown in FIG. 4, spinning and drawn artificial fibroin fibers were produced. The spinning device used is the spinning device 10 shown in FIG. 4 in which the second undrawn yarn producing device (the second undrawn yarn producing device (the first bath) and the wet heat drawing device 3 (third bath) are further provided. Second bath). The dry-wet spinning conditions are as follows.
Extrusion nozzle diameter: 0.2 mm
Extrusion speed (discharge amount): See Table 3 Liquid and temperature in the first to third baths: See Table 3 Winding speed: See Table 3 Total draw ratio: See Table 3 Drying temperature: 60°C
Air gap length: See Table 3

[(3) Evaluation of artificial fibroin fiber]
(Evaluation of shrinkage)
The shrinkage rate of each of the artificial fibroin fibers obtained in Production Examples 1 to 19 was evaluated. That is, each artificial fibroin fiber has a shrinkage rate (hereinafter, may be referred to as “primary shrinkage rate”) when subjected to a shrinkage process 1 of contacting with water having a boiling point or less, and water having a boiling point or less. The shrinkage factor (hereinafter, also referred to as “secondary shrinkage factor”) in the case where the shrinking process 2 is performed, which is drying at room temperature after contacting with, was evaluated.

<Primary shrinkage>
A plurality of artificial fibroin fibers for testing, each having a length of 30 cm, were cut out from each of the wound artificial fibroin fibers obtained in Production Examples 1 to 19. The artificial fibroin fibers were bundled to obtain an artificial fibroin fiber bundle having a fineness of 150 denier. A 0.8 g lead weight was attached to each artificial fibroin fiber bundle, and in that state, each artificial fibroin fiber bundle was immersed in water at the temperatures shown in Tables 4 to 10 for 10 minutes (shrinkage processing 1). Then, the length of each artificial fibroin fiber bundle was measured in water. The measurement of the length of the artificial fibroin fiber bundle in water was performed with the 0.8 g lead weight attached to the artificial fibroin fiber bundle in order to eliminate the crimping of the artificial fibroin fiber bundle. Then, the primary shrinkage (%) of each artificial fibroin fiber was calculated according to the following formula I. In Formula I, L0 represents the length of the artificial fibroin fiber bundle before being subjected to the shrinking process (here, 30 cm), and Lw represents the length of the artificial fibroin fiber bundle after being subjected to the shrinking process 1.
Formula I: Shrinkage rate (primary shrinkage rate)={1-(Lw/L0)}×100(%)

<Secondary shrinkage>
After immersion in water for evaluation of primary shrinkage, the artificial fibroin fiber bundle was taken out of water. The taken-out artificial fibroin fiber bundle was dried at room temperature for 2 hours with the 0.8 g lead weight attached (shrinkage process 2). After drying, the length of each artificial fibroin fiber bundle was measured. Next, the secondary shrinkage rate (%) of each artificial fibroin fiber was calculated according to the following formula II. In Formula II, L0 represents the length (here, 30 cm) of the artificial fibroin fiber bundle before shrinkage treatment, and Lwd represents the length of the artificial fibroin fiber bundle subjected to shrinkage treatment 2.
Formula II: Shrinkage rate (secondary shrinkage rate)={1-(Lwd/L0)}×100(%)

The results are shown in Tables 4-7.

From the above results, it is clearly recognized that the artificial fibroin fiber according to the present invention has a sufficiently high shrinkage ratio.

DESCRIPTION OF SYMBOLS 1... Extrusion device, 2... Undrawn yarn manufacturing device, 3... Wet heat drawing device, 4... Drying device, 6... Dope liquid, 10... Spinning device, 20... Coagulating liquid tank, 21... Drawing bath, 36... Artificial fibroin fiber , 38... High shrinkage artificial fibroin fiber, 40... Device, 42... Feed roller, 44... Winder, 46... Water bath, 48... Dryer, 54... Heater, 56... Tension roller, 58... Hot roller, 60... Processing device , 62... Drying device, 64... Dry heat plate.

Claims (4)

Man-made fibroin fiber containing modified fibroin and having a shrinkage ratio defined by the following formula of more than 7%.
Shrinkage rate={1-(length of artificial fibroin fiber that has been subjected to shrinkage processing including contact with water having a boiling point lower than that/length of artificial fibroin fiber before the shrinkage processing)}×100 (%) The artificial fibroin fiber according to claim 1, wherein the modified fibroin is a modified spider silk fibroin. The artificial fibroin fiber according to claim 1 or 2, wherein the temperature of the water is 10 to 90°C. The artificial fibroin fiber according to any one of claims 1 to 3, wherein the shrinking process includes drying after contact with the water.