JP2020020070A - Protein fiber and method for manufacturing the same - Google Patents

Abstract

To provide a novel artificial protein fiber that can easily adjust desired physical properties (strength and elongation, for example).SOLUTION: A composite fiber includes a first part including modified fibroin and a second part including structural protein that are linked in the fiber axis direction.SELECTED DRAWING: None

Description

  The present invention relates to a protein fiber and a method for producing the same.

  BACKGROUND ART Natural protein fibers such as silk and wool are widely used in materials such as clothing and bedding, taking advantage of their features such as excellent feel and heat retention. Natural protein fiber is biodegradable, and demand for a wider range of applications is expected in response to increasing awareness of environmental conservation.However, it is necessary to artificially control physical properties such as strength and elongation. There is a drawback that you can not.

  In recent years, researches for practical use of artificial protein fibers produced from modified fibroin obtained by modifying fibroin have been actively conducted (for example, see Patent Document 1). Such artificial protein fibers have higher strength and higher elongation than other protein fibers, and are expected to be applied not only to clothing but also to various fields such as industrial materials and medical materials. I have.

Japanese Patent No. 5540154

  However, even with such artificial protein fibers, it was not easy to adjust the strength and elongation to desired values.

  Therefore, an object of the present invention is to provide a new artificial protein fiber capable of easily adjusting desired physical properties (for example, strength and elongation). It is also an object of the present invention to provide a method for producing such a protein fiber.

The present invention provides the following [1] to [10].
[1]
A composite fiber in which a first portion containing a modified fibroin and a second portion containing a structural protein are joined in the fiber axis direction.
[2]
The composite fiber according to [1], wherein the structural protein is at least one selected from the group consisting of silk fibroin, spider silk fibroin, and keratin.
[3]
The composite fiber according to [1] or [2], wherein the thickness of the first portion and the thickness of the second portion are different from each other.
[4]
The conjugate fiber according to any one of [1] to [3], wherein a cross-sectional shape of the first portion and a cross-sectional shape of the second portion are different from each other.
[5]
Preparing a first dope containing the modified fibroin and the solvent, and a second dope containing the structural protein and the solvent;
Discharging one of the first dope solution and the second dope solution from the first spinneret;
Discharging the other dope liquid from the second spinneret so as to be continuous with the discharged dope liquid;
A step of putting the discharged continuous dope solution into a coagulation solution tank to obtain a fiber in which the first portion containing the modified fibroin and the second portion containing the structural protein are bonded in the fiber axis direction,
A method for producing a composite fiber, comprising:
[6]
The method according to [5], wherein the structural protein is at least one selected from the group consisting of silk fibroin, spider silk fibroin, collagen, resilin, elastin, and keratin.
[7]
The method for producing a conjugate fiber according to [5] or [6], wherein the diameter of the discharge hole of the first spinneret and the diameter of the discharge hole of the second spinneret are different from each other.
[8]
The method for producing a conjugate fiber according to any one of [5] to [7], wherein the shape of the discharge hole of the first spinneret and the shape of the discharge hole of the second spinneret are different from each other.
[9]
A composite fiber in which a first portion and a second portion are joined in a fiber axis direction,
When cut in a plane perpendicular to the fiber axis direction in the first portion, 95% or more of the cross-sectional area is composed of a site containing a modified fibroin, and 5% or less of the cross-sectional area is composed of a site containing a structural protein,
When cut at a plane perpendicular to the fiber axis direction at the second portion, a composite fiber comprising 95% or more of the cross-sectional area is composed of a site containing a structural protein and 5% or less of the cross-sectional area is composed of a site containing a modified fibroin. .
[10]
The composite fiber according to [9], wherein the structural protein is at least one selected from the group consisting of silk fibroin, spider silk fibroin, and keratin.

  According to the present invention, it is possible to provide a new artificial protein fiber capable of easily adjusting desired physical properties (for example, strength and elongation) and desired properties (for example, water shrinkability and crimpability).

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 derived from nature. It is a figure which shows the distribution of the value of x / y (%) of fibroin derived from nature. FIG. 2 is a schematic diagram showing a domain sequence of a modified fibroin according to one embodiment. FIG. 2 is a schematic diagram showing a domain sequence of a modified fibroin according to one embodiment. It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing a conjugate fiber. It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing a conjugate fiber. It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing a conjugate fiber. It is a schematic diagram of the conjugate fiber concerning one embodiment of the present invention. It is a schematic diagram of the conjugate fiber concerning one embodiment of the present invention. It is a schematic diagram of the conjugate fiber concerning one embodiment of the present invention.

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

  In the conjugate fiber according to the first embodiment of the present invention, the first part containing the modified fibroin and the second part containing the structural protein are joined in the direction of the fiber axis (the central axis of the conjugate fiber) to form one fiber. The fibers are formed. That is, the conjugate fiber according to the present embodiment exhibits the first portion exhibiting the physical properties and properties (functions) of the fiber containing the modified fibroin, and the physical properties and properties (function) of the fiber containing the structural protein. And a second portion are arranged side by side in the fiber axis direction. Thereby, in the composite fiber according to the present embodiment, the physical properties and characteristics having both the properties and properties derived from the modified fibroin fiber and the structural protein fiber, or the physical properties and properties derived from the modified fibroin fiber and the physical properties derived from the structural protein fiber New physical properties and characteristics, such as a fusion of properties and properties, can be exhibited. Further, in such a conjugate fiber, for example, the length of each of the first portion and the second portion is adjusted, or the type of the modified fibroin or the structural protein constituting each portion is changed or appropriately combined. By doing so, the physical properties and characteristics of the entire conjugate fiber can be arbitrarily controlled. In addition, in the conjugate fiber according to the present embodiment, even when the second portion contains the same modified fibroin as the first portion, for example, the thickness and the cross-sectional shape of the first portion and the second portion When the modified fibroin fibers have the same thickness and the same cross-sectional shape over the entire length, physical properties and characteristics that cannot be obtained are exhibited. In addition, physical properties and characteristics can be arbitrarily controlled by appropriately adjusting the thickness and the cross-sectional shape.

  Such composite fibers include, for example, (i) [first portion]-[second portion], (ii) [first portion]-[second portion]-[first portion], (Iii) The pattern [second part]-[first part]-[second part] may be joined in a state where it is repeated only once or plural times. That is, in the conjugate fiber according to the present embodiment, only one first portion and one second portion may be present, or at least one of the first portion and the second portion may be plural. May be present.

  In the conjugate fiber according to the present embodiment, when there are a plurality of first portions, each first portion may contain modified fibroin different from each other. When there are a plurality of second portions, each second portion may contain a different structural protein. Such conjugate fibers include, for example, (iv) [first portion A]-[second portion]-[first portion B], (v) [first portion A]-[first portion]. It may be joined in a pattern such as [part B]-[second part].

  The modified fibroin contained in the first portion of the conjugate fiber according to the present embodiment may have a property of contracting when brought into contact with an aqueous medium described below (hereinafter, also referred to as “water shrinkability”). .

(Modified fibroin)
The modified fibroin is a protein containing a domain sequence represented by 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, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.

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 naturally occurring fibroin, or may be the same as the amino acid sequence of naturally occurring fibroin. The term “naturally-derived fibroin” as used herein is also represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. Is a protein containing a domain sequence.

  `` Modified fibroin '' may be one using the amino acid sequence of naturally occurring fibroin as long as it has the amino acid sequence specified in the present invention, depending 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 a cloned naturally-derived fibroin), or may be artificially designed and used without depending on the naturally-derived fibroin. It may be a synthesized one (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 unique to fibroin (typically, corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence). The amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif Means Here, the (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (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. . (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, and is 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). At least seven of the (A) _n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP 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. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

  The modified fibroin is obtained by, for example, modifying the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to the cloned gene sequence of naturally occurring fibroin. Can be obtained at Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), and Methods in Enzymology, 100, 448 (1983).

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

  Examples of the fibroin produced by insects include Bombyx mori, Bombyx mandarina, natural silkworm (Antheraea yamamai), tussah (Anterea perinai), and maple silkworm (Eriogania seriatum). ), Silkworms produced by silkworms (Samia cynthia), chestnut worms (Caligura japonica), tussah silkworms (Antheraea mylitta), silkworms (Antherea assama), etc., and silkworms produced by the silkworm larvae (Vespa serrata) produced by the silkworm larvae (Vespa serrata). Silk proteins.

  More specific examples of fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), AAA27840.1 (amino acid sequence)).

  Examples of the fibroin produced by the spiders include spiders belonging to the genus Araneus (genus Araneus) such as the spiders, the spiders, the red spider, the red spider, and the green spider; And spiders belonging to the genus Pronus, such as spiders belonging to the genus Prosperum, and spiders belonging to the genus Pronus, and spiders belonging to the genus Cynotarachne, such as the spiders belonging to the genus Cyrtarachne, such as Torinofundamashi and Otorinofundamashi. (Spiders belonging to the genus Gasteracantha), spiders belonging to the genus Ordarius, such as spiders belonging to the genus Oryx argentina, and spiders belonging to the genus Ordgarius; Spiders belonging to the genus Argiope (Argiope spp.), Such as the Argiope spider, spiders belonging to the genus Arachnula (Arachnura), such as the arachnid spider, spiders belonging to the genus Acusilas (such as the genus Acusilas), such as the spiders of the spider spider and the spider spider spiders, such as the spider spider, the spiders Spiders belonging to the genus Spider belonging to the genus Cytophora, such as spiders and spiders belonging to the genus (Potys), spiders belonging to the genus Poltys, spiders belonging to the spider, spiders spiders, spiders belonging to the genus Cyclosa, spiders belonging to the genus Cyclos sp. Spider silk proteins produced by spiders belonging to the genus Genus), as well as red-eared spiders such as Red-headed Spider, Pleurotus guttata, Halabiro-ashida-gugu and Urocore-Pinegrass Spiders belonging to the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Nephla, such as spiders belonging to the genus Leucauge, such as spiders belonging to the genus Leucauge and spiders belonging to the genus Nephila, such as the spider Spiders belonging to the genus Dyschiriognatha such as spiders belonging to the genus Menosira and spiders belonging to the genus Dyschiriognatha, such as spiders belonging to the genus Dyschiriognatha, spiders belonging to the genus Latus sp. A spider produced by a spider belonging to the family Tetragnathidae, such as a spider belonging to the genus Prostenops. Pider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

  More specific examples of fibroin produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number 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, amino acid sequence from GenBank50 accession number (GenBank A4 accession number) U37520 (base sequence)), major ampullate spidroin 1 [Latrod ctus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [from Nephila clavata] (GenBank accession number AAL32472, 45 base sequence, amino acid sequence) major ampullate spidroin 1 [from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampullate spidroin 2 [Euprosanthos generos accessoline genera] Nos. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor amplify silkbank [Nonamp; Accession number AAC14591.1 (amino acid sequence)), minor ampli- nate spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence)) and the like.

  More specific examples of naturally occurring fibroin include fibroin in which sequence information is registered in NCBI GenBank. For example, among the sequence information registered in the NCBI GenBank, among sequences including INV as DIVISION, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION. It can be confirmed by extracting a character string of a specific product from a sequence or CDS and extracting a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

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

Specific examples of the modified fibroin include modified fibroin (first modified fibroin) derived from a large spinal canal thread protein produced in a spider's large ampullate line, modified fibroin having a reduced content of glycine residues. (second modified fibroin), (a) modified fibroin content of _n motifs has been reduced (third modified fibroin), the content of glycine residues, and the content of (a) _n motifs has been reduced Modified fibroin (fourth modified fibroin), modified fibroin having a domain sequence locally including a region with a large hydrophobicity index (fifth modified fibroin), and domain sequence having a reduced glutamine residue content Modified fibroin (sixth modified fibroin).

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

The first modified fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m and has a C-terminal sequence represented by any of SEQ ID NOS: 1 to 3 or The polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one 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 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is The amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.

  As a more specific example of the first modified fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1) or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroin comprising an amino acid sequence having at least% sequence identity. The sequence identity is preferably 95% or more.

  The amino acid sequence represented by SEQ ID NO: 4 is an amino acid sequence of ADF3 to which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site at the N-terminus is added. The 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue. The amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.

  The modified fibroin of (1-i) may be composed of the amino acid sequence represented by SEQ ID NO: 4.

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

  The second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine, as compared with a naturally-derived fibroin. At least one motif sequence selected from the group consisting of at least one glycine residue in one or more of the motif sequences has been 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 of the entire motif sequence.

The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the (A) _n motif located at the most C-terminal side from the domain sequence, to the domain sequence 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 up to the C-terminal of From, 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 to the C-terminal of the domain sequence is defined as 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 relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).

  The second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by replacing 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, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX below.

The method of calculating z / w will be described in more detail. First, in a fibroin (modified fibroin or naturally-occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , (A) _n located closest to the C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (there is no duplication), z is 50 × 3 = 150. Further, for example, when there is an X (center X) included in two XGXs as in the case of an amino acid sequence consisting of XGXGX, calculation is performed by subtracting 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 (the (A) _n motif located at the most C-terminal side is excluded). Next, 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 the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Of all the extracted fibroins, a naturally-derived one containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m and having an amino acid sequence consisting of GGX in the fibroin of 6% or less Z / w was calculated from the amino acid sequence of fibroin by the above-described calculation method. The result is shown in FIG. The horizontal axis in FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z / w of the naturally derived fibroin is less than 50.9% (the highest one is 50.86%).

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

  The second modified fibroin is modified, for example, by replacing at least a part of the base sequence encoding a glycine residue from the cloned natural fibroin gene sequence to encode another amino acid residue. Obtainable. At this time, as the glycine residue to be modified, a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more. Alternatively, for example, the amino acid sequence can be obtained by designing an amino acid sequence satisfying the above aspect 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 another amino acid residue for the glycine residue in the REP 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 insertion, addition, and / or addition may be modified.

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

  As more specific examples of the second modified fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met -PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence 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 represented by SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. The amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In this figure, one [(A) _n motif-REP] is inserted. The amino acid sequence represented by SEQ ID NO: 8 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: 7, and further has a partial glutamine (Q) residue. It is a substitution of a serine (S) residue and deletion of some C-terminal amino acids. The amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). Is obtained by adding Hinge and His tags to the C-terminal of the sequence obtained by repeating the above four times.

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

  The modified fibroin of (2-i) may be composed 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) includes 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. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The above 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 contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.

  The second modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.

  Examples of the tag sequence include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. 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 arranged and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including a His tag sequence and a hinge sequence).

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

  Further, an “epitope tag” utilizing an antigen-antibody reaction can 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 an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag. By using the epitope tag, the modified fibroin can be easily purified with high specificity.

  Furthermore, those in which the tag sequence can be cleaved by a specific protease can also be used. By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified fibroin from which the tag sequence has been separated can also be recovered.

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

  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. An amino acid sequence represented by SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) is added to the N-terminal of the amino acid sequence shown.

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

The modified fibroin of (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The above 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: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.

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

The third modified fibroin 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 third 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-derived fibroin.

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

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

The third modified fibroin has a domain sequence deletion of at least two (A) _n motifs from the N-terminal side to the C-terminal side, and one (A) It may have an amino acid sequence corresponding to the deletion of the _n motif repeated in this order.

The third modified fibroin may have a domain sequence having an amino acid sequence corresponding to the deletion of the (A) _n motif at least every third sequence from the N-terminal side to the C-terminal side. .

The third modified fibroin comprises 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] The number of amino acid residues of REP of the unit is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to When the maximum value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units of 11.3 is x, and the total number of amino acid residues in the domain sequence is y. 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 relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).

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 the modified fibroin. From the N-terminal side (left side), the domain sequence is (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) _It has a sequence called an _n motif.

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

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

In FIG. 1, when the smaller number of amino acid residues is set to 1, the [(A) _n motif-REP] unit pair in which the ratio of the other amino acid residues is 1.8 to 11.3 Shown by solid line. In the present specification, this ratio is called a jagged ratio. Assuming that the smaller number of amino acid residues is 1, the set of [(A) _n motif-REP] units in which the ratio of the other amino acid residues is less than 1.8 or more than 11.3 is indicated by a broken line. Indicated.

In each pattern, the total number of amino acid residues of two adjacent [(A) _n motif-REP] units shown by a solid line is added together (not only the REP but also the number of amino acid residues of the (A) _n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is 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 at least 50%, more preferably at least 60%, further preferably at least 65%, and even more preferably at least 70%. Preferably, it is still more preferably at least 75%, particularly preferably at least 80%. The upper limit of x / y is not particularly limited, and may be, for example, 100% or less. When the knurl ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more. When the knurl ratio is 1: 1.8 to 3.4, x / y is x / y. / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9% 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 seven of the (A) _n motifs present in a plurality in the domain sequence are composed of only alanine residues, x / y should be 46.4% or more. Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferred that there is. 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 the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Among all the extracted fibroins, x / y was calculated from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by Formula 1: [(A) _n motif-REP] _m by the above-described calculation method. Was calculated. FIG. 3 shows the results when the knurl ratio is 1: 1.9 to 4.1.

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

The third modified fibroin, for example, deletes one or more of the sequence encoding the (A) _n motif from the cloned natural fibroin gene sequence such that x / y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to the deletion of one or more (A) _n motifs is designed from the amino acid sequence of naturally occurring fibroin so that x / y is 64.2% or more, and 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 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 more specific examples of the third modified fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 represented by SEQ ID NO: 17 differs from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin in that every two amino acids from the N-terminal side to the C-terminal side (A) _n The motif was deleted, and one [(A) _n motif-REP] was inserted before the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.

  The x / y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) at a giza ratio of 1: 1.8 to 11.3 is 15.0%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 17 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.

  The modified fibroin of (3-i) may be composed 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) includes 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 Formula 1: [(A) _n motif-REP] _m . The above sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and is N-terminal to C-terminal. , 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 set to 1, the other And the amino acid residues of two adjacent [(A) _n motif-REP] units whose ratio of the number of amino acid residues of REP is 1.8 to 11.3 (giza ratio is 1: 1.8 to 11.3). It is preferable that x / y be 64.2% or more, where x is the maximum value of the sum of the base numbers and y is the total number of amino acid residues in the domain sequence.

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

  As more specific examples of the modified fibroin containing a tag sequence, (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) Modified fibroin comprising a sequence or (3-iv) an amino acid sequence having 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 can be mentioned. .

  The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding SEQ ID NO: 11 to the N-terminal 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. (Including a His tag sequence and a hinge sequence).

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

The modified fibroin of (3-iv) includes an amino acid sequence having 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. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The above 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. , 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 set to 1, the other Is the maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) _n motif-REP] units whose ratio of the number of amino acid residues of REP is 1.8 to 11.3. Preferably, x / y is 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

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

The fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motif and a reduced content of glycine residue as compared with a naturally derived fibroin. Have The domain sequence of the fourth modified fibroin differs from that of naturally occurring fibroin in that at least one or more (A) _n motifs have been deleted and at least one or more glycine residues in the REP have It can be said that it has an amino acid sequence equivalent to being replaced with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the second modified fibroin and the third modified fibroin described above. Specific embodiments and the like are as described for the second modified fibroin and the third modified fibroin.

  As a more specific example 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 And a modified fibroin comprising 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 represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 are as described above.

  The fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP have been replaced by amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and / or It may have an amino acid sequence locally including a region having a large hydrophobicity index, corresponding to insertion of one or more amino acid residues having a large hydrophobicity index therein.

  It is preferable that the region having a locally large hydrophobicity index is composed of continuous 2 to 4 amino acid residues.

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

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

  The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned natural fibroin gene sequence, It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. In addition, for example, one or more hydrophilic amino acid residues in REP were 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 REP. Can be obtained by designing an amino acid sequence corresponding to the insertion of the amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more hydrophilic amino acid residues in REP were 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 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 comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and extends from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence. In all REPs included in the sequence excluding the sequence from the domain sequence, the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p, Assuming that the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, p / q is 6 .2% or more.

  Regarding the hydrophobicity index of amino acid residues, a publicly known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydrodynamic character of a protein, B.Mol., J. Mol. 105-132). Specifically, the hydropathic index (hydropathic 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, a 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 Formula 1: [(A) _n motif-REP] _m (Hereinafter, referred to as “sequence A”). First, in all REPs included in sequence A, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of 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 four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.

For example, when “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” are extracted at 20 locations (without duplication), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2 The region of .6 or more would contain 20 consecutive 4 amino acid residues (no duplication), and p would be 20 × 4 = 80. For example, when two “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of four consecutive amino acid residues 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 duplication). For example, in the case of the domain sequence shown in FIG. 4, there are seven consecutive “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without overlapping. 28. Also, 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 (the last (A) _n motif on the C-terminal side is not included). 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 a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the cloned natural fibroin is satisfied so as to satisfy the above-mentioned p / q conditions. Substituting a negative amino acid residue with a hydrophobic amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can also be obtained by designing an amino acid sequence satisfying the above-mentioned 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 have been replaced by amino acid residues with a higher hydrophobicity index and / or one or more amino acid residues in the REP as compared to naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, a modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues may be performed. .

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

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

  The modified fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 19 is the same as the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) except for the terminal at the C-terminal side, which comprises three amino acid residues every other REP ( VLI) was inserted at two positions, a part of the glutamine residue (G) was replaced with a serine residue (S), and a part of the C-terminal amino acid was deleted. The amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). is there. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8.

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

The modified fibroin of (5-ii) contains an amino acid sequence having 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. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The above 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 at the most C-terminal side (A) _n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.

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

  As more specific examples of the modified fibroin including the tag sequence, (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) ) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

  The amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 correspond to the amino acid sequence represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).

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

The modified fibroin of (5-iv) contains an amino acid sequence having 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. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The above 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 at the most C-terminal side (A) _n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.

  The fifth modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the 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 has at least one motif selected from a GGX motif and a GPGXX motif in the amino acid sequence of REP.

  When the sixth modified fibroin contains a GPGXX motif in the REP, the content of the GPGXX motif is usually 1% or more, and may be 5% or more, and 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: fibroin containing a domain sequence represented by [(A) _n motif-REP] _m- (A) _n motif (modified fibroin or naturally occurring fibroin) Fibroin), the number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total number by 3 (ie, the total number of G and P in the GPGXX motif) is represented by s, and the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is represented by (A) When the total number of amino acid residues of all REPs excluding the (A) _n motif is represented by t, the content of the GPGXX motif is calculated as s / t. It is.

In the calculation of the content rate of the GPGXX motif, the target of “the sequence in which the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is “the most C-terminal side”. (A) Sequence from the _n motif to the C-terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of fibroin, and m may be small. In this case (that is, when the domain sequence is short), the calculation result of the GPGXX motif content is affected, so that this effect is eliminated. When the “GPGXX motif” is located at the C-terminus of the REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.

FIG. 5 is a schematic diagram showing the domain sequence of a modified fibroin. The method of calculating the content rate of the GPGXX motif will be specifically 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 at the most C-terminal side. (A) A sequence obtained by removing the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence ”(the sequence shown as“ region A ”in FIG. 5). 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 at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” (the sequence shown as “region A” in FIG. 5). ), The total number t of amino acid residues of all REPs excluding the (A) _n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, by dividing s by t, s / t (%) can be calculated. In the case of the modified fibroin of FIG. 5, 21/150 = 14.0%.

  The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more 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: fibroin containing a domain sequence represented by [(A) _n motif-REP] _m- (A) _n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the total number of glutamine residues contained in the region is defined as u, and the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence, and further (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 in which the sequence from the (A) _n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.

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

  The “other amino acid residue” may be any amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a 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). it can. Among them, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

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

In the present specification, the “REP hydrophobicity” is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: fibroin containing a domain sequence represented by [(A) _n motif-REP] _m- (A) _n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is defined as v, and the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence. A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding _n motifs. In the calculation of the degree of hydrophobicity of the REP, the reason why the “sequence in which the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.

  The sixth modified fibroin may have a domain sequence that is missing one or more glutamine residues in the REP and / or one or more glutamine residues in the REP, as compared to the naturally occurring fibroin. In addition to the modification corresponding to the substitution of with another amino acid residue, there may be an amino acid sequence modification equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues. .

  The sixth modified fibroin may, for example, delete one or more glutamine residues in the REP from the cloned naturally occurring fibroin gene sequence and / or remove one or more glutamine residues in the REP. By substituting the amino acid residue with In addition, 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 the above 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), modified fibroin comprising an amino acid sequence represented by SEQ ID NO: 29 (Met-PRT699), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698) or SEQ ID NO: 32 (Met-PRT1009), or (6-ii) having 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 Modified fibroin containing the amino acid sequence having the same.

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

  The amino acid sequence represented by SEQ ID NO: 30 is obtained by replacing all QQs in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence represented by SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 8 with VL and substituting the remaining Q with I.

  The amino acid sequence represented by SEQ ID NO: 32 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) (however, several amino acid residues on the C-terminal side of the region are substituted) Is repeated four times, all QQ in the sequence are replaced with VF, and the remaining Q is replaced with I.

  The amino acid sequences 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, and SEQ ID NO: 32 all have a glutamine residue content of 9% or less. (Table 2).

  The modified fibroin of (6-i) consists 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 There may be.

The modified fibroin of (6-ii) has 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 And amino acid sequences having the same sequence identity. The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. A protein containing a sequence. The above 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 include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.

  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 comprising the amino acid sequence represented by SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698) or SEQ ID NO: 40 (PRT1009), 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 a modified fibroin having an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 40.

  The amino acid sequences 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, and SEQ ID NO: 40 are SEQ ID NO: 25, SEQ ID NO: 26, 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 were added with the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) at the N-terminus. Things. Since only a tag sequence was added to the N-terminus, there was no change in the glutamine residue content, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 And the amino acid sequence represented by SEQ ID NO: 40 has a glutamine residue content of 9% or less (Table 3).

  The modified fibroin of (6-iii) consists 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 There may be.

The modified fibroin of (6-iv) has 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 And amino acid sequences having the same sequence identity. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. A protein containing a sequence. The above 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 include a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

  The structural protein contained in the second component may be a synthetic fiber or fibroin.

  Synthetic fibers generally have poor wettability and poor water absorbency and moisture absorbency. Examples of the synthetic fiber include polyester such as polyethylene terephthalate, polyamide such as polycaproamide (nylon 6) and nylon 66, polyacryl, and polyvinyl formal (vinylon). Synthetic fibers generally have the property of not easily shrinking even when brought into contact with an aqueous medium.

  The structural protein is not particularly limited, and may be produced by a microorganism or the like by genetic recombination technology, or may be produced by synthesis. Alternatively, the structural protein may be one obtained by purifying a naturally occurring structural protein.

  The structural protein may be, for example, a structural protein and an artificial structural protein derived from the structural protein. The term “structural protein” means a structural protein that forms or retains a structure and morphology in a living body. That is, the structural protein may be a naturally occurring structural protein, and a modified protein obtained by modifying a part of the amino acid sequence (for example, 10% or less of the amino acid sequence) based on the amino acid sequence of the naturally occurring structural protein It may be. Examples of the structural proteins include fibroin, keratin, collagen, elastin, and resilin.

  The fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin. In particular, the structural protein may be silk fibroin, spider silk fibroin or a combination thereof. When silk fibroin and spider silk fibroin are used in combination, the proportion of silk fibroin may be, for example, 40 parts by weight or less, 30 parts by weight or less, or 10 parts by weight or less based on 100 parts by weight of spider silk fibroin.

  The silk thread is a fiber (cocoon thread) obtained from a cocoon made by a silkworm, a larva of Bombyx mori. In general, one cocoon is composed of two silk fibroins and glue (sericin) covering them from outside. Silk fibroin is composed of a number of fibrils. Silk fibroin is covered with four layers of sericin. Practically, silk filaments obtained by dissolving and removing the outer sericin by scouring are used for clothing. A typical silk thread has a specific gravity of 1.33, a fineness of 3.3 decitex on average, and a fiber length of about 1300 to 1500 m. Silk fibroin is obtained using natural or silkworm cocoons or used or discarded silk fabric as a raw material.

  The silk fibroin may be a silk fibroin without sericin, a silk fibroin without sericin, or a combination thereof. Sericin-removed silk fibroin is purified by removing sericin covering silk fibroin and other fats. The silk fibroin purified in this manner is preferably used as a lyophilized powder. Silk fibroin without sericin is unpurified silk fibroin from which sericin and the like have not been removed.

  The spider silk fibroin may contain a spider silk polypeptide selected from the group consisting of a natural spider silk structural protein and a polypeptide derived from the natural spider silk structural protein (artificial spider silk structural protein).

  Examples of the natural spider silk structural protein include a large spinal cord marker silk structural protein, a weft protein, and a small ampullate gland structural protein. Since the large spinneret thread has a repeating region including a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity. The weft of spider silk has a feature that it does not have a crystalline region but has a repeating region composed of an amorphous region. The weft has a lower stress than the large spinneret and has a high elasticity.

  The large spinal cord marker thread structural protein is produced in the large ampullate gland of a spider and has a characteristic of being excellent in toughness. Examples of the large spinal cord marker thread structural protein include large ampullate gland spidroins MaSp1 and MaSp2 derived from the American spider (Nephila clavipes), and ADF3 and ADF4 derived from the Araneus diadematas. ADF3 is one of the two major bookmarker thread proteins of the Japanese spider. A polypeptide derived from a natural spider silk structural protein may be a polypeptide derived from these bookmarker silk structural proteins. ADF3-derived polypeptides are relatively easy to synthesize and have excellent properties in terms of strength and elongation and toughness.

  Weft thread structural proteins are produced in the flagellar gland of spiders. Examples of the weft thread structural protein include a flagellated silk structural protein derived from the American spider spider (Nephila clavipes).

  A polypeptide derived from a natural spider silk structural protein may be a recombinant spider silk structural protein. Examples of the recombinant spider silk structural protein include a mutant, analog or derivative of a natural spider silk structural protein. A preferred example of such a polypeptide is a recombinant spider silk structural protein of a large spider canal thread protein (also referred to as a “polypeptide derived from a large splenic canal thread structural protein”).

Examples of the fibroin-like structural protein derived from the large spinal cord tracing thread and the silkworm silk-derived structural protein include, for example, a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP1] _m Is mentioned. Here, in Formula 1, A of the (A) _n motif represents an alanine residue, and n is preferably an integer of 2 to 27, and is 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to It may be an integer of 16, 10 to 16, and (A) the number of alanine residues relative 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 , 90% or more and 100% (meaning that it is composed of only alanine residues). REP1 shows an amino acid sequence composed of 10 to 200 amino acid residues. m shows the integer of 10-300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REP1s may have the same amino acid sequence or different amino acid sequences.

In the above, a structural protein which has improved industrial productivity while maintaining strength and elongation by deleting the (A) _n motif in Formula 1 may be used. The frequency of deletion may be determined, for example, by sequentially comparing the number of REP amino acid residues in two adjacent [(A) _n motif-REP1] units from the N-terminal side to the C-terminal side. Assuming that the number of amino acid residues in the REP having a small number is 1, the two adjacent [(A) _n motif-REP1] units in which the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 X is the maximum value of the total value obtained by adding the number of amino acid residues, and y is the total number of amino acid residues in the domain sequence.

  Further, a structural protein having an amino acid sequence in which the content of glycine residues is reduced, which corresponds to at least one or more glycine residues in REP in REP of formula 1 being replaced with another amino acid residue. May be. As such a structural protein, there is a structural protein in which the ratio of the motif sequence in which a glycine residue is replaced with another amino acid residue is 10% or more of the entire motif sequence.

  Specific examples of the structural proteins derived from the large spinal cord guideline include structural proteins containing the amino acid sequences represented by SEQ ID NO: 13 and SEQ ID NO: 15.

Examples of the structural protein derived from the weft yarn structural protein include, for example, a structural protein containing a domain sequence represented by Formula 2: [REP2] _o (wherein, in Formula 2, REP2 is from Gly-Pro-Gly-Gly-X Wherein X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr) and valine (Val), and o represents an integer of 8 to 300. .). Specific examples include a structural protein comprising the amino acid sequence represented by SEQ ID NO: 13. The amino acid sequence represented by SEQ ID NO: 41 (PRT215) corresponds to the repeat portion and motif of the partial sequence of the flagellar silk structure protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAF36090, GI: 7106224) The amino acid sequence from the 1220th residue to the 1659th residue from the N-terminus (referred to as PR1 sequence) and the partial sequence of the flagellar silk structure protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAC38847; GI: 2833649) by combining the C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminal, and the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminal of the combined sequence. With the addition That.

As a collagen-derived structural protein, for example, a structural protein containing a domain sequence represented by Formula 3: [REP3] _p (where p represents an integer of 5 to 300 in Formula 3, and REP3 is Gly-X X and Y represent any amino acid residue except Gly. A plurality of REP3s may have the same amino acid sequence or different amino acid sequences.) Can be. Specifically, a structural protein containing the amino acid sequence represented by SEQ ID NO: 42 can be mentioned. The amino acid sequence represented by SEQ ID NO: 42 corresponds to a repeat portion and a motif of a partial sequence of human collagen type 4 obtained from the NCBI database (Accession number of GenBank of NCBI: CAA56335.1, GI: 3702452). The amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from residues 301 to 540.

As a resilin-derived structural protein, for example, a structural protein containing a domain sequence represented by Formula 4: [REP4] _q (where, in Formula 4, q represents an integer of 4 to 300. REP4 is Ser-J- 1 shows an amino acid sequence composed of J-Tyr-Gly-U-Pro, wherein J represents an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. Represents an arbitrary amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser .. A plurality of REP4s may have the same amino acid sequence or different amino acid sequences. Good)). Specifically, a structural protein containing the amino acid sequence represented by SEQ ID NO: 43 can be mentioned. In the amino acid sequence represented by SEQ ID NO: 43, in the amino acid sequence of resilin (NCBI GenBank Accession No. NP 611157, Gl: 246654243), Thr at the 87th residue was replaced with Ser, and Asn at the 95th residue was replaced with Ser. In which the amino acid sequence represented by SEQ ID NO: 11 (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 by Asp.

  Examples of elastin-derived structural proteins include structural proteins having an amino acid sequence such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specific examples include a structural protein containing the amino acid sequence represented by SEQ ID NO: 44. The amino acid sequence represented by SEQ ID NO: 44 corresponds to the amino acid sequence represented by SEQ ID NO: 11 (tag sequence) at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of GenBank Accession No. AAC98395 of NCBI. And a hinge sequence).

  Examples of the keratin-derived structural protein include Capra hircus type I keratin. Specifically, a structural protein containing the amino acid sequence represented by SEQ ID NO: 45 can be mentioned. The amino acid sequence represented by SEQ ID NO: 45 is obtained by adding the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequence of GenBank Accession No. ACY30466 of NCBI.

  One of the structural proteins or structural proteins derived therefrom can be used alone or in combination of two or more. The hydrophobicity as a whole may be adjusted to a desired value by combining two or more structural proteins.

  FIG. 9 is a schematic diagram of the conjugate fiber 10 according to one embodiment of the present invention. In the composite fiber 10 shown in FIG. 9, three first portions 1 and two second portions 2 are joined alternately, and the fiber diameter is uniform over the fiber length. The first portion comprises a modified fibroin having water shrinkage and can shrink upon contact with an aqueous medium. On the other hand, the second portion has reduced water shrinkage, and the shrinkage due to contact with water is smaller than that of the first portion. By appropriately changing the combination of the fibers used for the first component and the second component, or the composition ratio of the first component and the second component, the strength, elongation, water shrinkage, or the fiber when water shrinks as a whole It is possible to prepare a conjugate fiber having a desired value or degree of crimpability or the like generated by crimping. That is, these properties can be arbitrarily controlled in the composite fiber.

  Due to the difference in water shrinkage between the first component and the second component, when the conjugate fiber according to the present embodiment is brought into contact with an aqueous medium, the first portion shrinks more than the second portion, and is easily crimped. Processing and spinning can be performed. That is, the conjugate fiber according to the present embodiment has a crimping process as compared to a case where mechanical crimping or the like is performed while maintaining the characteristics of the second component, as compared with a fiber composed of only the second component. Sufficient crimpability can be obtained while advantageously reducing simplification and suppressing equipment costs, and can be useful as crimped yarn and spun yarn. The crimped yarn is excellent in spinnability, bulkiness, elasticity, flexibility, and elasticity, and can impart good texture, soft touch, and moisture retention. In addition, it is of course possible to employ a structural protein having greater water shrinkage than the modified fibroin constituting the first component as the second component.

  The hydrophobicity of the modified fibroin contained in the first component and the hydrophobicity of the structural protein contained in the second component are different from each other. The degree of hydrophobicity of the modified fibroin or structural protein is calculated by calculating the total of each HI of each amino acid residue (excluding the amino acid residues corresponding to the tag sequence and the hinge sequence), and calculating the total amino acid residue. It is the value divided by the radix. The hydrophobicity of the modified fibroin contained in the first component may be, for example, −0.8 or less, or −0.55 or less. The hydrophobicity of the structural protein contained in the second component may be, for example, more than -0.8 or more than -0.55.

  When the first component contains a plurality of modified fibroins, the degree of hydrophobicity of the component is calculated as the average value corresponding to the ratio of each component by calculating each degree of hydrophobicity of each modified fibroin contained in the first component. May be. For example, a value obtained by multiplying the degree of hydrophobicity of each modified fibroin by the content of the modified fibroin in the first component and summing the resulting values and dividing by the number of modified fibroins may be used.

  When the second component contains a plurality of structural proteins, the degree of hydrophobicity of the component is calculated as the average value corresponding to the ratio of each component by calculating each degree of hydrophobicity of each structural protein contained in the second component. May be. For example, a value obtained by multiplying the degree of hydrophobicity of each structural protein by the content of the structural protein in the second component, summing the obtained values, and dividing by the number of structural proteins may be used.

  In addition, as described above, when the first component contains a plurality of modified fibroins, or when the second component contains a plurality of structural proteins, the modified fibroin or the content is small (for example, the content is 10% or less). Since the structural protein has a sufficiently small contribution to the hydrophobicity of the entire component, the hydrophobicity of each component may be calculated without considering the modified fibroin or the hydrophobicity of the structural protein. For example, silk is composed of about 75% silk fibroin and about 25% sericin (UniProt database, Entry No. P07856). Silk fibroin is composed of fibroin heavy chain (UniProt database, Entry No. P05790), fibroin light chain (UniProt database, Entry No. P21828), and fibrohexamerin (UniProt database, Entry No. P04148). The fibroin heavy chain occupies the majority. When using silk fibroin of silkworm from which sericin has been removed as a natural structural protein, the hydrophobicity of the silk fibroin is calculated by calculating the sum of each HI of each amino acid residue of the main chain fibroin H chain, May be divided by the number of amino acid residues, and the hydrophobicity may be 0.216.

SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, 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 The HI is as shown in Table 4. In calculating the HI of each amino acid sequence, the HI was calculated excluding the sequence unrelated to the modified fibroin (that is, the sequence corresponding to the amino acid sequence represented by SEQ ID NO: 11).

HI of the amino acid sequences represented by SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45 are as shown in Table 5. In calculating the HI of each amino acid sequence, the HI was calculated excluding the sequence unrelated to the structural protein (that is, the sequence corresponding to the amino acid sequence represented by SEQ ID NO: 11).

  FIG. 10A is a schematic diagram of a conjugate fiber 20 according to another embodiment of the present invention. In the composite fiber 20 shown in FIG. 10A, three first portions 1 and two second portions 2 are joined alternately, and the fiber diameter of the second portion 2 is the first portion. 1 is smaller than the fiber diameter. As described above, the fiber diameter may be different for each part of the conjugate fiber. The strength, elongation, water shrinkage, crimpability, and the like can be arbitrarily controlled by reducing the diameter of a part of the conjugate fiber. Such a composite fiber 20 may be useful as a crimped yarn or a spun yarn, for example, if high crimpability is imparted.

  FIG. 10B is a schematic diagram of a conjugate fiber 30 according to another embodiment of the present invention. In the composite fiber 30 shown in FIG. 10B, the cross-sectional shape of the first portion 1 is circular, and the cross-sectional shape of the second portion 2 is triangular. The cross-sectional shape of the conjugate fiber can be appropriately changed depending on the shape of the discharge hole (hole) of the spinneret. FIG. 10C is a schematic cross-sectional view taken along line AA in FIG. 10B, and FIG. 10D is a schematic cross-sectional view taken along line BB in FIG. . As described above, the cross-sectional shape may be different for each part of the conjugate fiber. When such cross-sectional shapes differ from part to part, the joint is typically a combination of geometric features of adjacent cross-sectional shapes. For example, when a certain composite fiber has a form in which a portion having a circular cross-section and a portion having a triangular cross-section are joined, the joint may have a triangular shape with acute angles and / or rounded sides. . When the cross-sectional shapes of some of the conjugate fibers are different, for example, the touch and the like can be improved. When such a composite fiber 30 is used as a spun yarn or the like, there is obtained an advantage that, for example, the entanglement property is improved as compared with a fiber having a circular cross-sectional shape over its entire length.

  FIG. 11 is a schematic diagram showing a bonding surface of the conjugate fiber. FIG. 11A is a schematic diagram of a conjugate fiber 40 in which a first portion 1 and a second portion 2 are joined on a plane orthogonal to the central axis direction of the conjugate fiber. FIG. 11B is a schematic diagram of the conjugate fiber 50 in which the bonding surface has an elliptical shape inclined with respect to the central axis direction of the conjugate fiber. FIG. 11C is a schematic diagram of a conjugate fiber 60 in which the bonding surface has an irregular corrugated irregular shape. When the second component is ejected from the second spinneret while the first component is ejected from the first spinneret to form a joint region of both components, usually, as shown in FIG. 11B or 11C. It can be a good bonding surface. As the area of the bonding surface increases, the bonding energy of the first component and the second component increases, and the composite fiber can be bonded more firmly. Further, as shown in FIG. 11D, the first portion may include the second component as long as the physical properties of the first component are not suppressed. Further, the second component may include the first component as long as the physical properties of the second component are not suppressed. In such a conjugate fiber 70 in which the first component and the second component are mixed, the area occupied by the main component (for example, the first component) in the cross-sectional shape of the conjugate fiber in a plane orthogonal to the central axis is set. Preferably, it is 95% or more, 96% or more, 97% or more or 99% or more of the cross-sectional area of the conjugate fiber. If the area occupied by the main component (for example, the first component) in the cut shape is 95% or more, it is less likely to be affected by the physical properties of other components (for example, the second component).

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

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

  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, and the like), and can be appropriately selected depending on the type of the host. An inducible promoter that functions in a host cell and is capable of inducing the expression of a modified fibroin may be used as the promoter. 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 a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.

  The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. As the expression vector, those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed are suitably used.

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

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

  When a prokaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.

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

  When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEp13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, and competent methods.

  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, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .

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

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

  The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolyzate, 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 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, soybean meal hydrolyzate, various fermented cells and digests thereof can be used. As the inorganic salts, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

  Cultivation of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be performed under aerobic conditions such as, for example, shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culturing time is generally 16 hours to 7 days. The pH of the culture medium during the 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.

  During the culturing, if necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. When culturing a microorganism transformed with an expression vector using the trp promoter, indole acryl is used. An acid or the like may be added to the 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 the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton. The host cells are crushed with a Gaulin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for the isolation and purification of modified fibroin, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, an organic solvent , Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), and a resin such as S-Sepharose FF (manufactured by Pharmacia). Electrophoresis such as cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and isoelectric focusing Using methods such as the law alone or in combination, A purified sample can be obtained.

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

<Dope liquid>
The first dope solution contains a modified fibroin and a solvent. The second dope contains a structural protein and a solvent.

  The concentration of the modified fibroin in the first dope solution is not particularly limited, and may be appropriately set depending on factors such as the desired crimpability and fiber diameter of the conjugate fiber and the combination with the structural protein contained in the second component. Can be. For example, the concentration of the modified fibroin is preferably 5 to 40% by mass, based on the total mass of the first dope solution (when the total mass of the first dope solution is 100% by mass), and is preferably 7 to 40% by mass. , More preferably 10 to 40% by mass, more preferably 7 to 35% by mass, more preferably 10 to 35% by mass, and 12 to 35% by mass. More preferably, it is more preferably 15 to 35% by mass, still more preferably 15 to 30% by mass, further preferably 20 to 35% by mass, and more preferably 20 to 30% by mass. More preferred. When the concentration of the modified fibroin is 5% by mass or more, the productivity of the composite fiber tends to be further improved. When the concentration of the modified fibroin is 40% by mass or less, the dope solution can be more stably discharged from the spinneret, and the productivity tends to be further improved.

  The concentration of the structural protein in the second dope is not particularly limited, and is appropriately set depending on factors such as characteristics to be adjusted in a desired conjugate fiber, fiber diameter, and combination with the structural protein contained in the first component. can do. For example, the concentration of the modified fibroin is preferably 5 to 40% by mass, based on the total mass of the second dope solution (when the total mass of the second dope solution is 100% by mass), and is preferably 7 to 40% by mass. , More preferably 10 to 40% by mass, more preferably 7 to 35% by mass, more preferably 10 to 35% by mass, and 12 to 35% by mass. More preferably, it is more preferably 15 to 35% by mass, still more preferably 15 to 30% by mass, further preferably 20 to 35% by mass, and more preferably 20 to 30% by mass. More preferred. When the concentration of the modified fibroin is 5% by mass or more, the productivity of the composite fiber tends to be further improved. When the concentration of the modified fibroin is 40% by mass or less, the dope solution can be more stably discharged from the spinneret, and the productivity tends to be further improved.

  The solvent of the first dope solution may be any solvent that can dissolve the modified fibroin. The solvent of the second dope solution may be any solvent that can dissolve the structural protein. Such solvents include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), 1,3 -Dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine-N-oxide (NMO), formic acid and the like. Further, the solvent may be an aqueous solution, and specifically, an aqueous solution containing at least one selected from the group consisting of urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride and lithium thiocyanate. No. One of these solvents may be used alone, or two or more thereof may be used in combination.

  The method for preparing the dope solution may be a method well known to those skilled in the art, and the modified fibroin or structural protein and the solvent may be mixed in any order.

  An inorganic salt may be added to the first dope solution and the second dope solution as needed. The inorganic salt can function as a dissolution promoter for the modified fibroin. Examples of the inorganic salt include an alkali metal halide, an alkaline earth metal halide, an alkaline earth metal nitrate, and the like. Specific examples of inorganic salts include lithium carbonate, lithium chloride, calcium chloride, calcium nitrate, lithium bromide, barium bromide, calcium bromide, barium chlorate, sodium perchlorate, lithium perchlorate, and barium perchlorate. , Calcium perchlorate and magnesium perchlorate.

  The viscosity of the dope solution can be appropriately set according to the use of the conjugate fiber and the spinning method. The viscosity of the dope solution may be, for example, at 20 ° C., 5000 to 40000 mPa · sec, 7000 to 40000 mPa · sec, 10,000 to 40000 mPa · sec, 7000 to 35000 mPa · sec, 10,000 to 35000 mPa · sec, 10,000 to 30000 mPa · sec. sec or 10000-25000 mPa · sec. The viscosity of the dope solution can be measured using, for example, a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.

  The dope solution may be agitated or shaken for some time to facilitate dissolution. At that time, heating may be performed to a temperature at which the modified fibroin or the structural protein is dissolved in the solvent. The dope solution may be heated to, for example, 30 ° C. or higher, 40 ° C. or higher, 50 ° C. or higher, 60 ° C. or higher, 70 ° C. or higher, 80 ° C. or higher, or 90 ° C. or higher. The upper limit of the heating temperature is, for example, equal to or lower than the boiling point of the solvent.

<Spinning>
The conjugate fiber can be manufactured by a known spinning method. For example, the conjugate fiber can be obtained by spinning using a first dope solution and a second dope solution by a known spinning method such as dry spinning, melt spinning, wet spinning, and dry-wet spinning. Preferred spinning methods include wet spinning and dry spinning.

  In wet spinning or dry-wet spinning, a first dope solution and a second dope solution are discharged from a spinneret (nozzle) and joined, and a first component (modified fibroin) and a second component (structural protein) are coagulated in a coagulation solution. By hardening, a conjugate fiber can be obtained in an undrawn yarn state.

  FIG. 6 is an explanatory diagram schematically showing an example of a spinning device for producing a conjugate fiber. The spinning device 10 shown in FIG. 6 is an example of a spinning device for dry-wet spinning, and includes an extruder 1, an undrawn yarn manufacturing device 2, a wet heat drawing device 3, and a drying device 4.

  A spinning method using the spinning device 10 will be described. First, the dope solution 6 stored in the storage tank 7 is pushed out of the base 9 by the gear pump 8. On a laboratory scale (lab scale), a dope solution may be filled in a cylinder and extruded from a nozzle using a syringe pump. Next, the extruded dope solution 6 is supplied into the coagulation solution 11 of the coagulation solution tank 20 through the air gap 19, the solvent is removed, the protein is coagulated, and a fibrous coagulate is formed. Next, the fibrous coagulated material is supplied into the hot water 12 in the stretching bath 21 and stretched. The stretching ratio is determined by the speed ratio between the supply nip roller 13 and the take-off nip roller 14. Thereafter, the drawn fibrous coagulated body is supplied to the drying device 4 and dried in the yarn path 22, so that the composite fiber 36 is obtained as the wound body 5. 18a to 18g are yarn guides.

  In the method for producing a conjugate fiber according to the present embodiment, for example, a first dope solution and a second dope solution are spun by using a spinning device having a plurality of spinnerets and a reserve tank arranged as shown in FIG. 7 or 8. Discharge is performed from the base (nozzle) at the same discharge speed. The discharge from each spinneret is performed alternately at predetermined time intervals. When switching the dope liquid to be discharged, both of the dope liquids may be discharged to form a joint. In this case, a joint as shown in FIG. 11B or 11C may be formed.

  FIGS. 7 and 8 are schematic views showing a modified example corresponding to a portion from the extrusion device of the spinning device to the undrawn yarn production device shown in FIG. In FIG. 7, the two extruders 1 are arranged such that the respective spinnerets are substantially in contact with each other. One extruder is filled with the first dope, and the other extruder is the second dope. Is filled. The dope liquid to be discharged can be switched by opening and closing the gear pump 8 included in each extrusion device at a desired timing. The two spinnerets are arranged so that the fluxes of the respective dope liquids discharged into the air can be connected to each other, and are supplied into the coagulating liquid of the coagulating liquid layer via an air gap. After the dope liquid is supplied into the coagulation liquid, an undrawn yarn of the conjugate fiber in which the first component and the second component are joined is formed. Thereafter, as shown in FIG. 6, the stretched fibrous coagulated material is supplied to the drying device 4 and dried in the yarn path 22, and the composite fiber 36 is obtained as the wound yarn 5.

  In FIG. 8, the two extruders 1 have respective spinnerets connected through a switching valve, one extruder is filled with a first dope liquid, and the other extruder is supplied with a second dope liquid. Is filled. In the apparatus shown in FIG. 8, it is possible to easily switch the dope liquid to be discharged by operating the switching valve. The respective dope liquids are connected to each other in the switching valve, and the dope liquid discharged from the switching valve is supplied into the coagulation liquid of the coagulation liquid layer through an air gap. After the dope liquid is supplied into the coagulation liquid, an undrawn yarn of the conjugate fiber in which the first component and the second component are joined is formed. Thereafter, as shown in FIG. 6, the stretched fibrous coagulated material is supplied to the drying device 4 and dried in the yarn path 22, and the composite fiber 36 is obtained as the wound yarn 5.

  As a spinneret (nozzle) for producing a conjugate fiber, for example, a nozzle for producing a conjugate fiber having a side-by-side structure may be used. In a nozzle for producing a conjugate fiber having a side-by-side structure, a first die for extruding a first dope liquid (corresponding to the first component of the conjugate fiber) is arranged at a central portion of the nozzle, and a second dope liquid is provided close to the first die. A second base for extruding (corresponding to the second component of the conjugate fiber) is arranged. The second base may be inclined toward the first base so that the second dope is combined with the flux from the first dope extruded from the first base. Preferably, the flux from the first die and the flux from the second die are parallel. The second base may be substantially in contact with the first base. Further, the nozzle may have a structure in which the first base and the second base are integrated, and the discharge hole for the first dope liquid and the discharge hole for the second dope liquid are separated by a partition. Each base is preferably designed so that the discharge amount is constant by controlling the size and temperature. The second dope liquid extruded from the second die is combined with the first dope liquid extruded from the first die, is integrated, and comes into contact with the coagulation liquid, so that the unstretched conjugate fiber according to the present embodiment is not drawn. Allow the yarn to form.

  The position of each die can be appropriately adjusted according to the spinning conditions such as the type of fiber raw material used, the viscosity of each dope solution, the extrusion speed, and the temperature.

  The coagulating liquid 11 may be any solution that can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulating liquid 11 may appropriately contain water. Other known conditions such as the temperature of the coagulating liquid 11, the extrusion speed (discharge amount) of the nozzle, the residence time of the dope liquid (fiber) extruded from the nozzle in the coagulating liquid 11, and the drawing speed of the undrawn yarn, etc. May be appropriately adopted. Stretching may be performed at each stage or at a specific stage as necessary. The coagulation liquid may be maintained at a low temperature in order to suppress evaporation of the lower alcohol, and may be taken off in an undrawn yarn state. Further, the undrawn yarn may be drawn in the coagulating liquid (pre-drawing).

  The undrawn yarn (or pre-drawn yarn) obtained by the above method can be in a drawn yarn (composite fiber) state by the drawing step. Examples of the stretching method include wet heat stretching and dry heat stretching.

  The wet heat stretching can be performed in warm water, a solution obtained by adding an organic solvent or the like to warm water, or during steam heating. The temperature may be, for example, 40 to 200 ° C, 50 to 180 ° C, 50 to 150 ° C, or 75 to 90 ° C. The stretching ratio in the wet heat stretching may be, for example, 1 to 30 times, 2 to 25 times, 2 to 20 times, or 2 times to the undrawn yarn (or pre-drawn yarn). It may be up to 15 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, or 2 to 4 times. However, the draw ratio is not limited as long as the desired properties such as fiber thickness and mechanical properties can be obtained.

  Dry heat drawing can be performed using a device such as a contact-type hot plate and a non-contact type furnace, but it is not particularly limited, and the fiber is heated to a predetermined temperature, and Any device can be used as long as it can stretch at a magnification. The temperature may be, for example, 100C to 270C, 140C to 230C, 140C to 200C, 160C to 200C, or 160C to 180C. ° C.

  The draw ratio in the dry heat drawing step may be, for example, 1 to 30 times, 2 to 30 times, or 2 to 20 times with respect to the undrawn yarn (or pre-drawn yarn). 3 to 15 times, preferably 3 to 10 times, more preferably 3 to 8 times, even more preferably 4 to 8 times. However, the draw ratio is not limited as long as the desired properties such as fiber thickness and mechanical properties can be obtained.

  In the stretching step, the wet heat stretching and the dry heat stretching may be performed individually, or may be performed in multiple stages or in combination. That is, as the stretching step, wet heat stretching is performed as the first step stretching, and dry heat stretching is performed as the second step stretching, or wet heat stretching is performed as the first step stretching, wet heat stretching is performed as the second step stretching, and further, the third step stretching is performed. The stretching may be performed by dry heat stretching, or may be performed by appropriately combining wet heat stretching and dry heat stretching.

  The lower limit of the final draw ratio of the conjugate fiber that has undergone the drawing step is preferably 1, 2, 3, 4, 5, or 6 times the undrawn yarn (or pre-drawn yarn). , 7 times, 8 times, or 9 times. The upper limit of the final draw ratio of the conjugate fiber that has undergone the drawing step may preferably be 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. . Also, for example, it may be 3 to 40 times, may be 3 to 30 times, may be 5 to 30 times, may be 5 to 20 times, may be 5 to 15 times, and may be 5 to 15 times. It may be up to 13 times.

  In the spinning step, the shape of the spinneret, the shape of the discharge holes (holes), the number of holes, and the like are not particularly specified, and can be appropriately selected according to the desired fiber diameter, the number of single yarns, and the like.

  Before or after drying, if necessary, an oil agent may be added to the undrawn yarn (or pre-drawn yarn) or drawn yarn for the purpose of imparting antistatic properties, convergence, lubricity and the like. The type of oil agent to be applied, the amount to be applied, and the like are not particularly limited, and can be appropriately adjusted in consideration of the use of the conjugate fiber, the handleability of the conjugate fiber, and the like.

  When the hole shape of the spinneret is circular, a hole diameter of 0.1 mm to 0.6 mm can be exemplified. When the hole diameter is 0.1 mm or more, pressure loss can be reduced, and equipment costs can be reduced. When the pore diameter is 0.6 mm or less, the stretching operation for reducing the fiber diameter can be omitted, and the possibility of tearing (stretch breakage) between discharge and take-up can be further reduced.

  The temperature at the time of passing through the spinneret and the temperature of the spinneret are not particularly limited, and may be appropriately adjusted depending on the concentration and viscosity of the dope solution to be used, the type of the solvent, and the like. The temperature of the spinneret is preferably from 30C to 100C from the viewpoint of preventing the modified fibroin and the structural protein from deteriorating. In addition, the temperature may be increased to a temperature lower than the boiling point of the solvent to be used, from the viewpoint of increasing the pressure increase due to the volatilization of the solvent, and further reducing the possibility of causing blockage in the pipe due to solidification of the dope solution. preferable. This improves the process stability.

  The method according to the present embodiment may further include a step of filtering the dope liquid before discharging the dope liquid (filtration step) and / or a step of defoaming the dope liquid before discharge (defoaming step).

  The crimping step is a step in which the composite fiber having latent crimping ability is brought into contact with an aqueous medium to be crimped (hereinafter, may be referred to as “water crimping”).

  By contacting with an aqueous medium, the composite modified fibroin fiber can be crimped regardless of external force. The aqueous medium is a liquid or gas (steam) medium containing water (including water vapor). The aqueous medium may be water or a mixture of water and a hydrophilic solvent. Examples of the hydrophilic solvent include volatile solvents such as ethanol and methanol and vapors thereof. The aqueous medium may be a liquid mixture of water and a volatile solvent such as ethanol or methanol, and is preferably water or a liquid mixture of water and ethanol. By using an aqueous medium containing a volatile solvent or a vapor thereof, the drying rate after water crimping can be improved, and a soft texture can be imparted to the finally obtained crimped staple.

  The ratio of water to the volatile solvent or its vapor is not particularly limited, and for example, the mass ratio of water: volatile solvent may be from 10:90 to 90:10. The water content is preferably 30% by mass or more, and may be 40% by mass or 50% by mass or more based on the total mass of the aqueous medium. When the aqueous medium is a liquid, it is preferable to disperse the oil agent in the aqueous medium. In this case, water crimping and oil application can be performed simultaneously. In addition, as the oil agent, for example, a known oil agent used for a general purpose such as an antistatic, a friction reducing, a flexibility imparting, or a process passing property or a functional imparting such as a water repellency imparting. Any of them can be used. The amount of the oil agent is not particularly limited, and may be, for example, 1 to 10% by mass or 2 to 5% by mass based on the total mass of the oil agent and the aqueous medium.

  The aqueous medium is preferably a liquid or a gas containing water (including water vapor) at 10 to 230 ° C. The temperature of the aqueous medium may be 10C or more, 25C or more, 40C or more, 60C or more, or 100C or more, and may be 230C or less, 120C or less, or 100C or less. More specifically, when the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably from 100 to 230 ° C, more preferably from 100 to 120 ° C. When the steam of the aqueous medium is 230 ° C. or lower, thermal denaturation of the conjugate fiber can be prevented. When the aqueous medium is a liquid, the temperature of the aqueous medium is preferably 10 ° C. or more, 25 ° C. or more, or 40 ° C. or more from the viewpoint of efficiently imparting crimp, and is 60 ° C. from the viewpoint of keeping the strength of the conjugate fiber high. C. or less is preferred.

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

  Examples of the method of contacting with the aqueous medium include a method of immersing the conjugate fiber in the aqueous medium, a method of spraying the conjugate fiber with steam of the aqueous medium, and a method of exposing the conjugate fiber to an environment filled with the aqueous medium steam. No. When the aqueous medium is steam, the contact of the aqueous medium with the composite fiber can be performed using a general steam setting device. Specific examples of the steam set device include a product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and a product name: EPS-400 (manufactured by Tsujii Dyeing Machinery Co., Ltd.). As a specific example of the method of crimping the conjugate fiber by the steam of the aqueous medium, while storing the conjugate fiber in a predetermined storage chamber, the steam of the aqueous medium is introduced into the storage chamber, and the temperature in the storage chamber is set to the predetermined temperature. (Eg, 100 ° C. to 230 ° C.) while contacting the composite fiber with steam.

  In the step of crimping the conjugate fiber by contact with the aqueous medium, preferably, no tension is applied to the conjugate fiber (no tension is applied in the fiber axis direction), or the conjugate fiber is applied by a predetermined size (fiber). (Tension in the axial direction by a predetermined amount). The degree of crimp can be controlled by adjusting the tensile force applied to the composite fiber at that time. As a method of adjusting the tensile force applied to the conjugate fiber, for example, a method of adjusting the load applied to the fibers by suspending various weights on the conjugate fibers, a state in which the fibers are loosened In addition to identifying both ends with a method, various methods of changing the amount of slack, winding the fiber around a wound body such as a paper tube or bobbin, and the winding force (clamping force on the paper tube or bobbin) at that time. A method of appropriately changing the value may be used.

  After the conjugate fiber is brought into contact with the aqueous medium, it may be further dried. The drying method is not particularly limited, and the drying may be natural drying, heat storm or hot roller drying. The drying temperature is not particularly limited, and may be, for example, 20 to 150 ° C, preferably 40 to 120 ° C, and more preferably 60 to 100 ° C.

  In the conjugate fiber according to the present embodiment, the first component containing the modified fibroin and the second component containing the structural protein may have different degrees of hydrophobicity.

  The difference in the degree of hydrophobicity between the first component and the second component (the difference between the degree of hydrophobicity of the modified fibroin and the degree of hydrophobicity of the structural protein) in the conjugate fiber can be appropriately selected, for example, according to the desired crimpability. Possible, may be 0.1 or more, may be 0.2 or more, may be 0.3 or more, may be 0.4 or more, and may be 0.5 or more. 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1.0 or more It may be 1.1 or more, 1.2 or more, or 1.3 or more. The greater the difference in the degree of hydrophobicity, the more stably the excellent latent crimpability can be imparted.

The first component and the second component of the conjugate fiber may have different shrinkage rates due to water shrinkage. For example, the modified fibroin used for the first component contracts upon contact with the aqueous medium, while the structural protein used for the second component does not contract upon contact with the aqueous medium, or The contraction rate may be made smaller in comparison.
Table 6 shows the shrinkage ratio of modified fibroin fibers obtained by spinning modified fibroin suitable as the modified fibroin according to the present embodiment under the same conditions with respect to an aqueous medium. The shrinkage was calculated by the following method.
<Shrinkage>
A plurality of modified fibroin fibers having a length of about 30 cm are bundled to form a fiber bundle having a fineness of 150 denier. A 0.8 g lead weight is attached to this fiber bundle, and the fiber bundle is immersed in water at 40 ° C. for 10 minutes to shrink the fiber bundle in this state, thereby removing shrinkage due to residual stress originating in the manufacturing process. The fiber bundle is taken out of the water and left at room temperature for 2 hours with a 0.8 g lead weight attached to dry. After drying, the length of the fiber bundle is measured. It is immersed again in water at 40 ° C. for 10 minutes to shrink, and the length of the fiber bundle is measured in water. These wet and dry operations are repeated at least three times, and the average length when _wet (L _wet ) and the average length when _dry (L _dry ) are determined. The contraction rate is calculated according to the following equation.
Formula: Shrinkage (%) = (1− (L _dry / L _wet )) × 100

  The composite ratio of the first component and the second component in the composite fiber is not particularly limited, and is appropriately set according to a combination of the first component and the second component, a desired crimpability, a composite form, and the like. I can do this. The composite ratio of the first component and the second component may be, for example, in the range of 90:10 to 10:90, 80:20 to 20:80, or 75:25 to 25 on a weight basis. : 75: 25: 35: 35: 65, 70: 30: 30: 70, 65: 35: 35: 65. , 65:35 to 45:55, and 60:40 to 40:60.

  The cross-sectional shape of each part of the conjugate fiber is not particularly limited, and may be any of a round cross-section, a triangular cross-section, a multi-lobal cross-section, a bar-shaped cross-section, a flat cross-section, and other known cross-sectional shapes. When emphasis is placed on the balance between shrinkage and texture, a cross-sectional shape such as a semicircular side pie side type having a round cross section or a daruma cross section is preferable.

  The fiber diameter of each portion of the conjugate fiber is not particularly limited and can be appropriately set depending on the application and the like, and may be, for example, 10 to 125 μm, 10 to 100 μm, It may be 80 μm, 10 to 60 μm, 10 to 40 μm, 10 to 35 μm, or 10 to 30 μm. When the fiber diameter is 125 μm or less, the rate of desolvation in the spinning step is unlikely to increase. When the fiber diameter is 10 μm or more, it becomes easy to obtain a conjugate fiber stably.

  The number of crimps when the conjugate fiber is crimped with water can be appropriately set according to the application and the like, and may be, for example, 5 crimps / 25 mm or more, and 10 crimps / 25 mm or more. The number of crimps may be 15 pieces / 25 mm or more, the number of crimps may be 20 pieces / 25 mm or more, and the number of crimps may be 25 pieces / 25 mm or more.

  The conjugate fiber may be further chemically cross-linked between polypeptide molecules in the conjugate fiber depending on the use and the like. Examples of the functional group that can be crosslinked include an amino group, a carboxyl group, a thiol group, and a hydroxy group.

Cross-linking between polypeptide molecules may be performed using a cross-linking agent such as carbodiimide or glutaraldehyde, or may be performed using an enzyme such as transglutaminase. Carbodiimide is represented by the general formula R ¹ NＮC ＝ NR ² (wherein R ¹ and R ² each independently represent an organic group containing an alkyl group having 1 to 6 carbon atoms and a cycloalkyl group). Compound. Specific examples of carbodiimide include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N′-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide , Diisopropylcarbodiimide (DIC) and the like. Among these, EDC and DIC are preferable because they have a high ability to form amide bonds between polypeptide molecules and easily undergo a crosslinking reaction.

  In the cross-linking treatment, it is preferable to apply a cross-linking agent to the conjugate fiber and cross-link by heating under vacuum. The cross-linking agent may be applied to the conjugate fiber as a pure product, or may be applied to the conjugate fiber after being diluted to a concentration of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms and a buffer. Good. The crosslinking treatment is preferably performed at a temperature of 20 to 45 ° C. for 3 to 42 hours. By the cross-linking treatment, a higher stress (strength) can be applied to the conjugate fiber.

(Evaluation of crimpability of composite fiber)
The crimpability of the conjugate fiber can be evaluated by confirming the number of crimps of the conjugate fiber that has developed a latent crimp through a crimping step.

[Product]
The conjugate fiber according to the present embodiment can be applied to a fabric such as a woven fabric, a knitted fabric, a braid, and a nonwoven fabric as a fiber or a yarn. Further, the conjugate fiber according to the present embodiment can be applied to long fibers, short fibers, multifilaments, spun yarns, twisted yarns, processed yarns, blended yarns, blended yarns, nonwoven fabrics, paper, and cotton. It can be manufactured according to the method described in Japanese Unexamined Patent Publication No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735, and the like.

  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 and Structural Protein (1) Preparation of Expression Vector The modified fibroin is based on the nucleotide sequence and amino acid sequence of fibroin (GenBank Accession No .: P468804.1, GI: 1174415) derived from Nephila clavipes. , SEQ ID NO: 15 (PRT799) and SEQ ID NO: 40 (PRT1009) were designed to have modified structural proteins (hereinafter, also referred to as “PRT799” and “PRT1009”, respectively). The amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues with respect to the amino acid sequence of fibroin derived from Nephila clapives for the purpose of improving productivity. Further, an amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminus. The amino acid sequence represented by SEQ ID NO: 40 (PRT1009) is an amino acid sequence represented by SEQ ID NO: 7 (an amino acid sequence before the amino acid sequence represented by SEQ ID NO: 11 is added to the N-terminus) for the purpose of improving hydrophobicity. In the region of the 20 domain sequences present therein, all the QQs in the sequence repeated four times were replaced with VF, the remaining Qs were replaced with I, and the N-terminal amino acid sequence represented by SEQ ID NO: 11 (tag Sequence and hinge sequence). Further, as a structural protein, a modified structural protein having an amino acid sequence represented by SEQ ID NO: 46 (hereinafter, also referred to as “PRT798”) based on the base sequence and the amino acid sequence of the structural protein of Caora hircus (GenBank Accession No .: NP001272643.1) ).

  Next, nucleic acids encoding PRT799 and PRT1009 (modified fibroin) and PRT798 (structural protein) were synthesized. An NdeI site at the 5 'end and an EcoRI site downstream of the stop codon were added to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and then recombined into the protein expression vector pET-22b (+) to obtain an expression vector.

(2) Expression of modified fibroin and structural protein Escherichia coli BLR (DE3) was transformed with the expression vector obtained in the above (1). 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 a seed culture medium containing ampicillin (Table 7) 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 seed culture solutions.

The seed culture solution was added to a jar fermenter to which 500 mL of a production medium (Table 8) had been added so that the OD ₆₀₀ was 0.05. The culture was maintained at a temperature of 37 ° C. and controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturated concentration.

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

(3) Purification of Modified Fibroin and Structural Protein Two hours after the addition of IPTG, the recovered cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 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 a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in an 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL. For 30 minutes with a stirrer 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 the dialysis was collected by centrifugation, the water was removed with a freeze dryer, and the freeze-dried powder was collected to obtain a modified fibroin (PRT799 and PRT1009) and a structural protein (PRT798).

2. Production of Silk Fibroin (Structural Protein) (1) Preparation of Silk Fibroin Powder Using a natural silkworm (Bombyx mori) cocoon, the silkworm cocoon from which the content was removed was cut into small pieces. After boiled for about 30 minutes in boiling 0.5% by mass Marcel soap water (Marcel soap used was fined with a grater), it was boiled in boiling water for 30 minutes. This procedure was repeated twice more (three times in total). Finally, the resultant was boiled with boiling water for 30 minutes to completely remove sericin covering the silk fibroin, and dried the silk fibroin after removing the sericin at 37 ° C. overnight. After drying, the silk was weighed, and an aqueous lithium bromide solution (9 mol / L) was added so as to have a concentration of 10% by mass / volume, followed by dissolving at 40 ° C. for 2 hours. This aqueous solution was put into a cellulose dialysis membrane (Seamless Cellulose Tubing, 36/32 manufactured by VISKASESELESCOAP) and dialyzed with distilled water for 3 to 4 days. The recovered solution after dialysis was centrifuged at 20 ° C. and 15000 rpm for 1 hour to remove undissolved and impurities. Further, it was diluted with Milli water so that the concentration became 2% by mass or less. After dilution, the solution was passed through a 150 μm filter manufactured by ADVANTEC to completely remove impurities. The silk fibroin aqueous solution was frozen at -80 ° C and freeze-dried overnight. After confirming that water was sufficiently removed, silk fibroin powder was obtained.

3. Production and evaluation of composite fiber <Example 1>
(1) Preparation of Dope Solution As a first component, 24% by mass of modified fibroin (PRT799) obtained in the process of producing the modified fibroin and 76% by mass of formic acid (98% purity) as a solvent were mixed and mixed at 40 ° C. The mixture was heated for 1 hour with an aluminum block heater to dissolve, filtered through a metal filter having a mesh size of 1 μm, and defoamed to prepare a first dope solution.
A second dope was prepared in the same manner as the first dope except that 24% by weight of silk fibroin obtained in the step of producing silk fibroin (structural protein) was used as the second component.

(2) Production of Composite Fiber Using the apparatus shown in FIG. 7, the first dope solution and the second dope solution were discharged from each spinneret (nozzle) at the same discharge speed. The dope discharged from each spinneret was switched at predetermined intervals. After alternately discharging the first dope liquid and the second dope liquid, known spinning, drawing, and drying steps were performed. When switching between the discharge of the first dope liquid and the discharge of the second dope liquid, both dope liquids were simultaneously discharged for a predetermined time.
In the obtained conjugate fiber, the first portion and the second portion were alternately joined in the fiber axis direction, and the joined portion had a form shown in FIG. 11B. This composite fiber had a lower tensile strength than the protein fiber produced only with the modified fibroin (PRT799).

  DESCRIPTION OF SYMBOLS 1 ... Extrusion apparatus, 2 ... Undrawn yarn manufacturing apparatus, 3 ... Wet heat drawing apparatus, 4 ... Drying apparatus, 6 ... Dope liquid, 9 ... Spinneret, 10 ... Spinning apparatus, 11 ... Coagulation liquid, 20 ... Coagulation liquid tank, 21 ... Drawing bath, 36 ... Composite fiber.

Claims (8)

  A composite fiber in which a first portion containing a modified fibroin and a second portion containing a structural protein are joined in the fiber axis direction.   The composite fiber according to claim 1, wherein the structural protein is at least one selected from the group consisting of silk fibroin, spider silk fibroin, and keratin.   The conjugate fiber according to claim 1, wherein a thickness of the first portion and a thickness of the second portion are different from each other.   The conjugate fiber according to any one of claims 1 to 3, wherein a cross-sectional shape of the first portion and a cross-sectional shape of the second portion are different from each other. Preparing a first dope solution containing the modified fibroin and the solvent, and a second dope solution containing the structural protein and the solvent;
Discharging one of the first dope solution and the second dope solution from the first spinneret;
Discharging the other dope liquid from the second spinneret so as to be continuous with the discharged dope liquid;
A step of putting the discharged continuous dope liquid into a coagulation bath to obtain a fiber in which the first portion containing the modified fibroin and the second portion containing the structural protein are bonded in the fiber axis direction,
A method for producing a conjugate fiber, comprising:   The method according to claim 5, wherein the structural protein is at least one selected from the group consisting of silk fibroin, spider silk fibroin, collagen, resilin, elastin, and keratin.   The method for producing a conjugate fiber according to claim 5, wherein a diameter of a discharge hole of the first spinneret and a diameter of a discharge hole of the second spinneret are different from each other. The method for producing a conjugate fiber according to any one of claims 5 to 7, wherein a shape of a discharge hole of the first spinneret and a shape of a discharge hole of the second spinneret are different from each other.

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