JPWO2018164189A1 - Protein molded article, method for producing the same, and protein solution - Google Patents

Abstract

A protein solution containing a protein, a reactive compound, and a solvent in which these are dissolved, wherein the reactive compound is a compound having a first functional group and a second functional group, and the first functional group is A group capable of binding a protein and a reactive compound by reaction with a protein, wherein the second functional group is a group capable of binding two or more reactive compounds to each other by a reaction of two or more second functional groups; A step of preparing a protein solution, a step of obtaining a protein-containing molded body by molding using the protein solution as a stock solution, and a step of subjecting the molded body to post-treatment for bonding two or more reactive compounds to each other And a method for producing a protein molded article, comprising the steps of:

Description

  The present invention relates to a protein molded product, a method for producing the same, and a protein solution.

  Protein molded products such as fibers and films sometimes have characteristics of easily shrinking. Shrinkage of a protein compact can cause various problems during its manufacturing or use. As a method for suppressing the shrinkage of a protein molded article, there have been proposed several methods of imparting a crosslinking agent to a molded article obtained by molding to crosslink proteins in the molded article (Patent Documents 1 to 3).

JP-A-10-310977 Japanese Patent No. 5540154 JP 2009-221616 A

  It is desirable that the shrinkage of the protein molded body be suppressed as small as possible. In particular, the protein molded article often has a property of relatively shrinking largely due to the initial contact with water after production, and further shrinking by subsequent drying, and it is desired to suppress such shrinkage.

  Accordingly, an object of one aspect of the present invention is to suppress shrinkage due to contact with moisture immediately after production and shrinkage due to drying of a protein molded article.

One aspect of the present invention provides a method for producing a protein molded body. This method
A protein solution containing a protein and a reactive compound and a solvent in which the protein and the reactive compound are dissolved, wherein the reactive compound has a first functional group and a second functional group different from the first functional group. Wherein the first functional group is a group capable of binding the protein to the reactive compound by reaction with the protein, and the second functional group is a group capable of binding two or more reactive compounds by the reaction of two or more second functional groups. Preparing a protein solution, which is a group capable of binding to each other,
A step of obtaining a protein-containing molded body by molding using a protein solution as a stock solution for molding,
Subjecting the molded article to a post-treatment for bonding two or more reactive compounds to each other by a reaction of two or more second functional groups.

  According to this method, it is possible to suppress shrinkage of the molded body due to contact with moisture immediately after molding and shrinkage of the molded body due to subsequent drying.

  Another aspect of the present invention provides a protein molded article containing a protein cross-linked by a reactive compound. The reactive compound is a compound having a first functional group and a second functional group different from the first functional group. The reaction between the first functional group and the protein causes the protein and the reactive compound to bind, and the reaction between the two or more second functional groups causes the two or more reactive compounds to bind to each other, whereby the protein is cross-linked. I have.

  This protein molded product is unlikely to cause shrinkage due to contact with water immediately after production and shrinkage due to subsequent drying.

  Yet another aspect of the present invention provides a protein solution comprising a protein and a reactive compound and a solvent in which they are dissolved. The reactive compound is a compound having a first functional group and a second functional group different from the first functional group. The first functional group is a group capable of binding a protein and a reactive compound by a reaction with a protein, and the second functional group is a group capable of binding two or more reactive compounds to each other by a reaction of two or more second functional groups. It is a group that can be used.

  By using this protein solution as a stock solution for molding, it is possible to suppress shrinkage of the molded body due to contact with moisture immediately after production and shrinkage of the molded body due to subsequent drying.

  ADVANTAGE OF THE INVENTION According to the method concerning one aspect of this invention, shrinkage | contraction of a protein molded object by contact with water | moisture content immediately after manufacture, and subsequent shrinkage of the protein molded object accompanying drying can be suppressed.

It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. It is the schematic which shows an example of the spinning apparatus for producing a protein fiber. It is a schematic diagram showing an example of a heating device for subjecting protein fiber to a heat treatment. ^{1 is} a ¹ H NMR spectrum of spider silk fibroin treated with a solution of a reactive compound. It is a ¹ H NMR spectrum by the DOSY method of the spider silk fibroin processed by the solution of the reactive compound. FIG. 7 is an enlarged view showing a region B in FIG. 6.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as necessary. However, the present invention is not limited to the following embodiments.

  One embodiment of a method for producing a protein molded article, a step of preparing a protein solution containing a protein and a reactive compound and a solvent in which these are dissolved, and molding using the protein solution as a stock solution for molding, The method may include a step of obtaining a molded article containing a protein and a step of subjecting the molded article to post-treatment such as heat treatment.

  The reactive compound may be a compound having a first functional group capable of binding the reactive compound and the protein and a second functional group capable of binding the reactive compounds. In this case, the post-treatment can be a treatment for bonding two or more reactive compounds to each other by a reaction of two or more second functional groups.

<Protein>
The protein constituting the protein molded body is not particularly limited, and may be a protein produced by a microorganism or the like by a genetic recombination technique or a protein produced by synthesis. Alternatively, the protein may be a protein obtained by purifying a naturally occurring protein.

  The protein may include, for example, a structural protein. The term “structural protein” means a protein that forms or maintains a structure, form, or the like in a living body. The structural protein may be an artificial structural protein derived from a natural structural protein. Structural proteins include, for example, fibroin, keratin, collagen, elastin and resilin.

  The structural protein may be fibroin. 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.

  A silk thread is a fiber (cocoon thread) obtained from a cocoon made by a silkworm, a larva of Bombyx mori. Generally, one cocoon thread is composed of two silk fibroins and glue (sericin) covering them from the 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 outer sericin by refining are used for clothing. A general 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 sericin-depleted silk fibroin, non-sericin-depleted silk fibroin, or a combination thereof. The sericin-removed silk fibroin is purified by removing sericin covering the silk fibroin and other fats. The silk fibroin thus purified may be a lyophilized powder. Silk fibroin without sericin is unpurified silk fibroin from which sericin or the like has not been removed.

  The spider silk fibroin may contain one or more spider silk polypeptides selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins (artificial spider silk proteins).

  Natural spider silk proteins include, for example, large spinal canal thread proteins, weft proteins, and small bottle gland proteins. Since the large spinneret thread has a repetitive 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 has no repetitive region consisting of an amorphous region without having a crystalline region. The weft has a lower stress but a higher elasticity as compared with the large spinneret.

  The large spinal canal thread protein is produced in a spider's large bottle-shaped line, and is characterized by excellent toughness. Examples of the large spinal cord bookmarker thread protein include large ampullate spidroins MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Araneus diadematus. ADF3 is one of the two major bookmarker thread proteins of the Japanese spider. Polypeptides derived from natural spider silk proteins may be polypeptides derived from these bookmarked silk proteins. ADF3-derived polypeptides are relatively easy to synthesize and have excellent properties in terms of strength and elongation and toughness.

  Weft protein is produced in the flagellar gland of spiders. As the weft protein, for example, flagellaform silk protein derived from Nephila clavipes is mentioned.

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

  Examples of the fibroin-like protein derived from a major spinal cord marker thread or a silkworm silk-derived protein include, for example, Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif- REP] m- (A) n. Here, (A) n motif is an amino acid sequence mainly containing an alanine residue, and n is an integer of 2 to 27. n may be an integer from 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 is 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more. % Or more, 95% or more, or 100% (meaning that the (A) n motif is composed of only alanine residues). At least seven of the multiple (A) n motifs present in the domain sequence represented by Formula 1 or 2 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. A plurality of (A) n motifs present in the domain sequence represented by Formula 1 or 2 may have the same amino acid sequence or different amino acid sequences. A plurality of REPs present in the domain sequence represented by Formula 1 or 2 may have the same amino acid sequence or different amino acid sequences. As a specific example of the protein derived from the large spinal cord marker thread, a protein containing the amino acid sequence represented by SEQ ID NO: 1 can be mentioned.

  The modified fibroin derived from the major spinal cord marker thread protein produced in the spider large ampullate contains a unit of the amino acid sequence (domain sequence) represented by Formula 1: [(A) n motif-REP] m. , As the C-terminal sequence, an amino acid sequence represented by any of SEQ ID NOs: 14, 15 or 16, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 14, 15 or 16. A polypeptide.

  The amino acid sequence shown in SEQ ID NO: 14 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). The amino acid sequence represented by SEQ ID NO: 15 is the same as the amino acid sequence represented by SEQ ID NO: 14 by removing 20 residues from the C-terminal. The amino acid sequence represented by SEQ ID NO: 16 is the same as the amino acid sequence represented by SEQ ID NO: 14 by removing 29 residues from the C-terminal.

  As a more specific example of the modified fibroin derived from the major spinal cord marker thread protein produced in the large ampullate of the spider, the amino acid sequence represented by (1-i) SEQ ID NO: 17 or the (1-ii) sequence Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 17 can be mentioned. The sequence identity here may be 95% or more.

  The amino acid sequence represented by SEQ ID NO: 17 is obtained by adding an amino acid sequence (SEQ ID NO: 18) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site to the N-terminus; This corresponds to a sequence mutated so that the number of the 13th repetitive region is increased about 2-fold and the translation is terminated at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17 is the same as the amino acid sequence represented by SEQ ID NO: 16.

  The modified fibroin of (1-i) may consist of the amino acid sequence represented by SEQ ID NO: 17.

  A modified fibroin having a reduced content of glycine residues has an amino acid sequence in its domain sequence in which the content of glycine residues is reduced as compared to naturally occurring fibroin. The modified fibroin can have an amino acid sequence corresponding to one in which at least one or more glycine residues in the REP have been replaced with another amino acid residue, as compared to naturally occurring fibroin.

  The domain sequence of the modified fibroin in which the content of the glycine residue is reduced is GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is glycine An amino acid residue selected from at least one motif sequence selected from the group consisting of at least one glycine residue and another amino acid residue in the motif sequence. It may be.

  In the modified fibroin in which the content of the glycine residue is reduced, the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence. .

The modified fibroin in which the content of the glycine residue is reduced contains the domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and is the most C-terminal of the domain sequences represented by Formula 1. In the sequence other than the portion from the (A) _n motif to the C-terminus of the relevant domain sequence, XGX (G is a glycine residue, X is an amino acid residue other than glycine) contained in all REPs. ), When the total number of amino acid residues is z and the total number of amino acid residues is w, the ratio of z to w (z / w,%) is 30% or more, 40% or more, 50% Or more than 50.9%. (A) The ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 83% or more, 86% or more, 90% or more, or 95% or more. The ratio may be 100% (meaning that the (A) _n motif is composed of only alanine residues).

  Modified fibroin with reduced glycine residue content is obtained by replacing one glycine residue of the GGX motif with another amino acid residue. The content of the amino acid sequence consisting of XGX may be increased. In the modified fibroin in which the content of glycine residues is reduced, the content of the amino acid sequence consisting of GGX in the domain sequence is 30% or less, 20% or less, 10% or less, 6% or less, 4% or less, or 2%. The following may be used. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX.

The method of calculating z / w will be described in more detail. First, in a fibroin containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m (modified fibroin or naturally-derived fibroin), the domain sequence located at the most C-terminal side (A) _An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence other than the portion from the _n motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting the extracted XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted without duplication, z is calculated as 50 × 3 = 150. For example, when there is an X included in two XGXs, such as a central X in an amino acid sequence consisting of XGXGX, the number of overlapping Xs is subtracted. That is, XGXGX is regarded as a sequence containing five amino acid residues constituting XGX. w is the total number of amino acid residues contained in the sequence other than the portion from the (A) _n motif located closest to the C-terminus to the C-terminus of the domain sequence in the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is calculated as 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230. In this calculation, four amino acid residues in the (A) _n motif located closest to the C-terminal are excluded. From z and w, the ratio of z to w (z / w,%) is calculated.

  In the modified fibroin having a reduced content of glycine residues, z / w may be 50.9% or more, 56.1% or more, 58.7% or more, 70% or more, or 80% or more. . The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

  Modified fibroin having a reduced content of glycine residues may be obtained by, for example, encoding at least a part of a nucleotide sequence encoding a glycine residue from a cloned natural fibroin gene sequence to encode another amino acid residue. It can be obtained by modifying 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 a base encoding the glycine residue may be selected so that z / w is 50.9% or more. The sequence may be replaced. For example, by designing an amino acid sequence that satisfies the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence, obtaining a modified fibroin with a reduced content of glycine residues You can also. 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 the addition, insertion 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 ) Residues, proline (P) residues, hydrophobic amino acid residues such as phenylalanine (F) residues and tryptophan (W) residues, or glutamine (Q) residues, asparagine (N) residues, serine ( It may be a hydrophilic amino acid residue such as S) residue, lysine (K) residue and glutamic acid (E) residue, and valine (V) residue, leucine (L) residue and isoleucine (I) residue. It may be a group or a glutamine (Q) residue or a glutamine (Q) residue.

  As more specific examples of the modified fibroin having a reduced content of glycine residues, (2-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2-i) ii) Modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 can be mentioned.

The modified fibroin (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 3 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin with GQX. The amino acid sequence represented by SEQ ID NO: 4 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: 3, and further before the C-terminal sequence. In which one [(A) _n motif-REP] was inserted. The amino acid sequence represented by SEQ ID NO: 10 has two alanine residues inserted at the C-terminal side of each (A) _n motif of the amino acid sequence represented by SEQ ID NO: 4, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence obtained by adding a His tag to the C-terminus of a sequence obtained by repeating the above four times.

  The value of z / w in the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally occurring fibroin) is 46.8%. The values of z / w in the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 10, and the amino acid sequence represented by SEQ ID NO: 12 are 58.7%, respectively. 70.1%, 66.1% and 70.0%. The values of x / y of the amino acid sequences represented by SEQ ID NOs: 1, 3, 4, 10, and 12 at the indentation ratio of 1: 1.8 to 11.3 are 15.0%, 15.0%, and 93. 4%, 92.7% and 89.3%. The details of the jaw ratio and x / y will be described later.

  The modified fibroin of (2-i) may be composed of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity may be 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: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and contains XGX ( Wherein G is a glycine residue and X is an amino acid residue other than glycine.) The total number of amino acid residues in the amino acid sequence consisting of z is z, and the total number of REP amino acids in the domain sequence represented by Formula 1 is z. When the number is w, the ratio of z to w (z / w,%) may be 50.9% or more.

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

  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: 5 (amino acid sequence including a His tag).

  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.

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

  A sequence that can cleave the tag sequence with 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 including the tag sequence, (2-iii) the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 8 , SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 13.

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

  The modified fibroin of (2-iii) may consist of the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

The modified fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity may be 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: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, and contains XGX ( Here, G represents a glycine residue and X represents an amino acid residue other than glycine.) The total number of amino acid residues in the amino acid sequence consisting of z is z, and the total number of amino acid residues of REP in the domain sequence is w. Sometimes, the ratio of z to w (z / w,%) may be 50.9% or more.

  The modified fibroin described above 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 host.

(A) modified fibroin content of _n motifs has been reduced, the domain sequence is compared to the naturally occurring fibroin, having an amino acid sequence reduced the content of (A) _n motif. The domain sequence of the modified fibroin can have an amino acid sequence corresponding to the deletion of at least one or more (A) _n motifs, as compared to a naturally occurring fibroin.

The modified fibroin in which the content of the (A) _n motif is reduced may have an amino acid sequence equivalent to 10 to 40% of the deletion of the (A) _n motif from a naturally occurring fibroin.

(A) The domain sequence of the modified fibroin in which the content of the _n motif is reduced is at least every 1 to 3 (A) _n motifs from the N-terminal side to the C-terminal side, as compared with the naturally-derived fibroin. The amino acid sequence may correspond to the deletion of one (A) _n motif.

(A) _n motif content domain sequence of reduced modified fibroin, as compared to the naturally occurring fibroin, two successive toward at least the N-terminal side to the C-terminal side of the (A) _n motif deleted It may be an amino acid sequence corresponding to the deletion and deletion of one (A) _n motif repeated in this order.

(A) The domain sequence of the modified fibroin in which the content of the _n motif has been reduced is 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. There may be.

(A) The modified fibroin in which the content of the _n motif is reduced may include a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In this case, the number of amino acid residues of the REP of two adjacent [(A) _n motif-REP] units is sequentially compared from the N-terminal side to the C-terminal side. Assuming that the number of amino acid residues is 1, the amino acid residues of two adjacent [(A) _n motif-REP] units such that the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3. When the maximum value of the sum of the numbers is x and the total number of amino acid residues in the domain sequence represented by Formula 1 is y, the ratio of x to y (x / y,%) is 20%. The above may be 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, 86% or more, 90% or more, or 95% or more, and 100% ((A) _n motif is alanine (Meaning that it is composed of only 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, an unselected [(A) _n motif-REP] unit may be present. 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 fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP with the fifth REP), and pattern 4 (the first REP and the fifth REP). Second REP comparison). The selection method is not limited to these.

Next, for each pattern, the number of amino acid residues of each REP in the selected two [(A) _n motif-REP] units adjacent to each other is compared. The ratio of the number of the other amino acid residues is determined. 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 The ratio of the number of amino acid residues is calculated as 100/50 = 2. Similarly, when comparing the fourth REP (20 amino acid residues) with 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 calculated as 30/20 = 1.5.

In FIG. 1, when the smaller number of amino acid residues is set to 1, the ratio of the other amino acid residues (hereinafter, sometimes referred to as “giza ratio”) is 1.8 to 11.3. A set of such [(A) _n motif-REP] units is shown by a solid line. A set of [(A) _n motif-REP] units whose giza ratio is less than 1.8 or more than 11.3 is indicated by a broken line.

In each pattern, summing up all the amino acid residues of the two [(A) _n motif -rep] units adjacent indicated by the solid line ((A) amino acid residues of _n motifs included). The total value of the total number of amino acid residues in each pattern is compared, and the total value of the total number of amino acid residues in the pattern having the maximum total value (the maximum value of the total value) is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

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

(A) In the modified fibroin in which the content of the _n motif is reduced, x / y may be 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more. . The upper limit of x / y is not particularly limited, and may be, for example, 100% or less. The knurl ratio may be 1: 1.9 to 11.3, x / y may be 89.6% or more, and the knurl ratio is 1: 1.8 to 3.4, and x / y is 77.1. % Or more, the knurl ratio may be 1: 1.9 to 8.4, x / y may be 75.9% or more, and the knurl ratio is 1: 1.9 to 4.1. , X / y may be 64.2% or more.

In the modified fibroin in which the content of the (A) _n motif is reduced, when at least seven (A) _n motifs present in the domain sequence are composed of only alanine residues, x / y is 46.4%. The above may be 50% or more, 55% or more, 60% or more, 70% or more, or 80% or more. The upper limit of x / y is not particularly limited, and may be 100% or less.

(A) The modified fibroin in which the content of the _n motif is reduced, for example, encodes the (A) _n motif so that x / y is 64.2% or more based on the gene sequence of the cloned natural fibroin. It can be obtained by deleting one or more of the sequences. For example, based on the amino acid sequence of naturally occurring fibroin, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed so that x / y is 64.2% or more, and the designed amino acid By chemically synthesizing a nucleic acid encoding the sequence, it is also possible to obtain a modified fibroin having a reduced content of the (A) _n motif. 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 modification may be modified.

(A) As more specific examples of the modified fibroin in which the content of the _n motif is reduced, (3-i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or 3-ii) Modified fibroin including an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12 can be mentioned.

The modified fibroin (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 2 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: 1 corresponding to naturally occurring fibroin. , And one [(A) _n motif-REP] inserted before the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 4 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 2 with GQX. The amino acid sequence represented by SEQ ID NO: 10 has two alanine residues inserted at the C-terminal side of each (A) _n motif of the amino acid sequence represented by SEQ ID NO: 4, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 12 corresponds to a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence obtained by adding a His tag to the C-terminal of a sequence repeated four times.

  The x / y value of the amino acid sequence represented by SEQ ID NO: 1 (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: 2 and the amino acid sequence represented by SEQ ID NO: 4 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 12 is 89.3%. The values of z / w in the amino acid sequences represented by SEQ ID NOs: 1, 2, 4, 10, and 12 were 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively. is there.

  The modified fibroin of (3-i) may be composed of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity may be 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: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and has a Giza ratio of 1: 1.8. The maximum value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units such that 11.3 is x, and the total number of amino acid residues in the domain sequence is y , The ratio of x to y (x / y,%) may be 64.2% or more.

  The modified fibroin described above 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 sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 13.

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

  The modified fibroin of (3-iii) may consist of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

The modified fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity may be 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: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, and has a Giza ratio of 1.8 to 11. The maximum value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units is x, and the total number of amino acid residues in the domain sequence is y. Sometimes, the ratio of x to y (x / y,%) may be 64.2% or more.

  The modified fibroin described above 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 host.

The content of glycine residues, and (A) modified fibroin content of _n motifs has been reduced, the domain sequence is compared to the naturally occurring fibroin was reduced content of (A) _n motif In addition, it has an amino acid sequence in which the content of glycine residues is reduced. The domain sequence of the modified fibroin differs from that of a naturally occurring fibroin in that at least one or more (A) _n motifs are deleted and at least one or more glycine residues in REP are different It has an amino acid sequence equivalent to being replaced with an amino acid residue. This modified fibroin is a modified fibroin having the characteristics of the modified fibroin having a reduced content of the glycine residue and the modified fibroin having a reduced content of the (A) _n motif. Specific embodiments and the like are as described in the modified fibroin in which the content of the glycine residue is reduced and the modified fibroin in which the content of the (A) _n motif is reduced.

As more specific examples of the modified fibroin in which the content of the glycine residue and (A) the _n motif are reduced, (4-i) an amino acid represented by SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12 Examples of the modified fibroin include an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by the sequence, (4-ii) SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12. Specific embodiments of the modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12 are as described above.

  In another embodiment, the modified fibroin has a domain sequence in which one or more amino acid residues in the REP have been replaced with amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin; and And / or may have an amino acid sequence locally including a region having a large hydrophobicity index, corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index into the REP.

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

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

  In the modified fibroin according to the present embodiment, one or more amino acid residues in REP have been replaced with amino acid residues having a large hydrophobicity index, and / or 1 Alternatively, in addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, one or more amino acid residues may be substituted, deleted, inserted and / or compared with a naturally occurring fibroin. Alternatively, there may be an amino acid sequence modification corresponding to the addition.

  The modified fibroin according to the present embodiment is, for example, one or more hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydrophobicity index) from the cloned gene sequence of naturally occurring fibroin. It can be obtained by substituting an amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. For example, replacing one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or inserting one or more hydrophobic amino acid residues into REP An altered fibroin 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. In any case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the REP. In addition to the modification corresponding to the insertion of a residue, the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The modified fibroin according to yet another embodiment includes a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and (A) _n located at the most C-terminal side of the domain sequence. In sequences other than the portion from the motif to the C-terminus of the domain sequence, the total number of amino acid residues contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues in all REPs is 2.6 or more is p When the total number of amino acid residues is q, the ratio of p to q (p / q) may be 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 hydrographic 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. In the calculation, the sequence other than the portion from the (A) _n motif located closest to the C-terminal to the C-terminal of the domain sequence in the domain sequence represented by Formula 1: [(A) _n motif-REP] _m is used. (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 determined for all four consecutive amino acid residues. Each amino acid residue is used for calculating an average value 1 to 4 times. Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even 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 2.6. The region described above includes 20 consecutive 4 amino acid residues (without duplication). p is calculated as 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 only one amino acid residue, the average of the consecutive four amino acid residues of the hydrophobicity index is A region having a value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “−1” is a subtraction of duplication). For example, in the case of the domain sequence shown in FIG. 2, there are seven consecutive “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without overlapping, so p is 7 × 4 = 28 is calculated. In the case of the domain sequence shown in FIG. 2, q is calculated as 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170. In this calculation, the number of amino acid residues in the last (A) _n motif on the C-terminal side is not included. By dividing p by q, the ratio of p to q (p / q,%) can be calculated. In the case of FIG. 2, p / q is calculated as 28/170 = 16.47%.

  In the modified fibroin according to the present embodiment, p / q may be 6.2% or more, 7% or more, 10% or more, 20% or more, or 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

  The modified fibroin according to the present embodiment may be obtained, for example, by changing the amino acid sequence of a cloned natural fibroin to one or more hydrophilic amino acid residues (for example, hydrophobic Replacing amino acid residues with a negative sex index with hydrophobic amino acid residues (eg, amino acid residues with a positive hydrophobic index) and / or one or more hydrophobic amino acid residues during REP Can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. For example, a modified fibroin can be obtained by designing an amino acid sequence that satisfies the above-mentioned p / q conditions from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP were replaced by amino acid residues having a large hydrophobicity index, and / or one or more amino acid residues in the REP were 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 the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. .

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

  As another specific example of the modified fibroin, (5-i) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 22, or (5-ii) SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 22 And a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by.

The modified fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 4 is obtained by deleting an amino acid sequence in which alanine residues are consecutive in the (A) _n motif of a naturally occurring fibroin so that the number of consecutive alanine residues becomes five. . The amino acid sequence represented by SEQ ID NO: 19 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues at every other REP with respect to the amino acid sequence represented by SEQ ID NO: 4; A part of the amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence becomes almost the same as that of the amino acid sequence. The amino acid sequence represented by SEQ ID NO: 20 is different from the amino acid sequence represented by SEQ ID NO: 19 in that two alanine residues are inserted at the C-terminal side of each (A) _n motif, and a part of glutamine (Q) residue This group is obtained by substituting a group with a serine (S) residue and deleting a part of the amino acids at the C-terminal side so that the molecular weight of the amino acid sequence shown in SEQ ID NO: 4 is almost the same. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP into the amino acid sequence represented by SEQ ID NO: 20. The amino acid sequence represented by SEQ ID NO: 22 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: 20.

  The modified fibroin of (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22.

The modified fibroin of (5-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity may be 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: 21 or SEQ ID NO: 22, and is the most C-terminal of the domain sequences. (A) Regarding the sequence other than the portion from the _n motif to the C-terminus of the domain sequence, the amino acid residue included in the region where the average value of the hydrophobicity index of four consecutive amino acid residues in all REPs is 2.6 or more. When the total number of groups is p and the total number of amino acid residues is q, the ratio of p to q (p / q,%) may be 6.2% or more.

  The modified fibroin described above 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 containing the tag sequence, (5-iii) the amino acid sequence represented by SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or (5-iv) SEQ ID NO: 23, SEQ ID NO: 24 or Modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25 can be mentioned.

  The amino acid sequences represented by SEQ ID NOS: 23, 24 and 25 are obtained by adding the amino acid sequence represented by SEQ ID NO: 5 (including the His tag sequence and the hinge sequence) to the N-terminal of the amino acid sequences represented by SEQ ID NOs: 19, 21 and 22, respectively. It has been added.

  The modified fibroin of (5-iii) may consist of the amino acid sequence represented by SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

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: 23, SEQ ID NO: 24 or SEQ ID NO: 25. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity may be 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: 23, SEQ ID NO: 24 or SEQ ID NO: 25, and is located closest to the C-terminal side of the domain sequence. (A) In sequences other than the portion from the _n motif to the C-terminus of the domain sequence, amino acid residues contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues in all REPs is 2.6 or more Is p and the total number of amino acid residues is q, the ratio of p to q (p / q,%) may be 6.2% or more.

  The modified fibroin described above 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 host.

Examples of the protein derived from the weft protein include, for example, a protein containing a domain sequence represented by Formula 3: [REP2] _o (wherein REP2 is an amino acid sequence composed of Gly-Pro-Gly-Gly-X 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. it can. Specific examples of the protein derived from the weft protein include a protein containing the amino acid sequence represented by SEQ ID NO: 26. The amino acid sequence represented by SEQ ID NO: 26 was obtained from the N-terminus corresponding to the repeat portion and the motif of the partial sequence of the flagellar silk protein of the American spider spider obtained from the NCBI database (NCBI accession numbers: AAF36090, GI: 7106224). The amino acid sequence from residues 1220 to 1659 (referred to as PR1 sequence) and the partial sequence of the flagellar silk protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649) A C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminus is joined, and the amino acid sequence represented by SEQ ID NO: 5 (tag sequence and hinge sequence) is added to the N-terminus of the joined sequence; is there.

As a collagen-derived protein, for example, a protein containing a domain sequence represented by Formula 4: [REP3] _p (in Formula 4, p represents an integer of 5 to 300. REP3 is composed of Gly-X-Y. X and Y each represent an amino acid residue other than Gly. A plurality of REP3s may have the same amino acid sequence or different amino acid sequences.) As a specific example of the protein derived from collagen, a protein containing the amino acid sequence represented by SEQ ID NO: 27 can be mentioned. The amino acid sequence represented by SEQ ID NO: 27 corresponds to the repeat portion and the motif of the 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: 5 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from residues 301 to 540.

As a protein derived from resilin, for example, a protein containing a domain sequence represented by Formula 5: [REP4] _q (in Formula 5, q represents an integer of 4 to 300. REP4 is Ser-J-J-Tyr-Gly 1 shows an amino acid sequence composed of one U-Pro, J represents an arbitrary amino acid residue, and in particular, may be an amino acid residue selected from the group consisting of Asp, Ser, and Thr. A REP4, which may be 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. be able to. As a specific example of the protein derived from resilin, a protein containing the amino acid sequence represented by SEQ ID NO: 28 can be mentioned. In the amino acid sequence represented by SEQ ID NO: 28, in the amino acid sequence of resilin (GenBank accession number of NCBI, Accession No. NP 611157, Gl: 246654243), Thr at residue 87 is replaced with Ser, and Asn at residue 95 is replaced with Ser. In which the amino acid sequence represented by SEQ ID NO: 5 (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 proteins include proteins having an amino acid sequence such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine), and the like. Specific examples of the elastin-derived protein include a protein containing the amino acid sequence represented by SEQ ID NO: 29. The amino acid sequence represented by SEQ ID NO: 5 corresponds to the amino acid sequence represented by SEQ ID NO: 5 (tag sequence) at the N-terminus of the amino acid sequence from residue 121 to residue 390 in the amino acid sequence of GenBank Accession No. AAC98395 of NCBI. And a hinge sequence).

  One of the structural proteins described above and proteins derived from the structural proteins can be used alone or in combination of two or more.

  The protein contained in the protein solution and the protein molded product is, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.

  The method for producing the nucleic acid encoding the protein is not particularly limited. For example, the nucleic acid can be produced by a method of amplifying and cloning by the polymerase chain reaction (PCR) using a gene encoding a natural structural protein, or a method of chemically synthesizing. The method for chemically synthesizing nucleic acids is not particularly limited. For example, AKTA oligopilot plus 10/100 (GE Healthcare Japan) based on amino acid sequence information of structural proteins obtained from the NCBI web database or the like. The gene can be chemically synthesized by a method of linking oligonucleotides automatically synthesized by the method such as PCR. At this time, in order to facilitate purification and / or confirmation of the protein, a nucleic acid encoding a protein consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence was synthesized. Is also good.

  The regulatory sequence is a sequence that controls the expression of the recombinant protein 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 target protein 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. The expression vector may be capable of autonomous replication in a host cell or may be integrated into a host chromosome, and may contain a promoter at a position where a nucleic acid encoding a protein of interest can be transcribed.

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

  Examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium or Pseudomonas. Examples of the microorganism 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. Examples of the microorganism belonging to the genus Bacillus include Bacillus subtilis. Examples of the microorganism belonging to the genus Microbacterium include 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 for introducing a nucleic acid encoding a target protein include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569).

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

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

  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 protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in the culture medium can be 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, as a culture medium, a medium containing a carbon source, a nitrogen source, inorganic salts, and the like that can be utilized by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.

  The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysate, and organic acids such as acetic acid and propionic acid. 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 fermentation cells and digests thereof can be used. As the inorganic salts, for example, potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

  Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during the culture may be kept 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 culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed. 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 protein can be isolated and purified by a commonly used method. For example, when the protein is expressed in a dissolved state in the cells, after culturing, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, and Manton Gaulin. The host cells are crushed with a homogenizer and a dynomill to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying proteins, that is, a solvent extraction method, a salting-out method with ammonium sulfate, a desalting method, a method using an organic solvent Precipitation method, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose FF (manufactured by Pharmacia). Electrophoresis methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Etc. are used alone or in combination. Goods can be obtained.

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

<Reactive compound>
The reactive compound according to one embodiment may be a compound having a first functional group capable of binding the reactive compound and the protein, and a second functional group capable of binding the reactive compounds. The reactive compound can function as a cross-linking agent that cross-links the protein molecular chain by the reaction between the first functional group and the second functional group.

  The first functional group of the reactive compound may be any functional group capable of reacting with a functional group of the protein, such as a carboxy group, amino group, hydroxy group, or thiol group, to form a bond. The first functional group is, for example, an isocyanate group, a blocked isocyanate group, a thiol group, an aldehyde group, an ester group, an epoxy group, an aziridine group, a chloro group, a bromo group, an iodo group, a carboxylic anhydride group, a chloroformyl group, It may be at least one group selected from the group consisting of an N-hydroxyphthalimide group, an N-hydroxysuccinimide group, a maleimide group, and a cyanuric chloride group. The number of the first functional groups included in the reactive compound may be one, or may be two or more.

  The second functional group of the reactive compound may be any functional group capable of forming a bond by a reaction between the second functional groups, and is usually different from the first functional group. The second functional group may be a radically polymerizable unsaturated group such as a vinyl group (for example, acryloyl group, methacryloyl group), and an alkynyl group (for example, ethynyl group). The number of the first functional groups included in the reactive compound may be one, or may be two or more.

  Specific examples of the reactive compound include 1,1- (bisacryloyloxymethyl) ethyl isocyanate (BEI), 2-methacryloyloxyethyl isocyanate (MOI), and 2-acryloyloxyethyl isocyanate (AOI). BEI has one isocyanate group as a first functional group and two acryloyl groups as a second functional group. MOI and AOI have one isocyanate group as a first functional group and one (meth) acryloyl group as a second functional group.

  As a first step, in a protein solution containing a protein and a reactive compound, at least a part of the reactive compound may bind to the protein by reacting the first functional group with a functional group in the protein. . For example, a reaction between an isocyanate group as a first functional group and a hydroxy group in a protein may generate a urethane bond, whereby the reactive compound may bind to the protein. Thereafter, as a second step, when the molded body obtained by molding using the protein solution as a stock solution for molding is subjected to a post-treatment (crosslinking treatment), at least a part of the reactive compound bound to the protein is: It is considered that the second functional groups are bonded to each other by a reaction between them. For example, the reactive compounds can be bonded to each other by a polymerization reaction of a radically polymerizable unsaturated group as the second functional group. As a result, it is considered that the molecular chains of the protein are cross-linked by the reactive compound. However, some of the second functional groups may have reacted at a stage before the post-treatment step, such as in a protein solution before being subjected to molding. Further, at the stage of the post-treatment process, the second functional groups may react with each other and the first functional groups may react with the protein.

  As described above, according to the method of progressively cross-linking a protein using a reactive compound having two or more kinds of functional groups, the reactive compound can penetrate deeply into the inside of the molded article. It is considered that the cross-linking between proteins proceeds more reliably and efficiently to the inside of the molded article, as compared with the method of cross-linking proteins by applying a cross-linking agent to the surface of the body. This stepwise crosslinking is thought to form a specific crosslinked structure inside the molded body, but quantitatively specifying that structure requires many trial and errors, and is not. However, it is speculated that the cross-linked structure formed through stepwise cross-linking is a factor that not only suppresses the shrinkage due to moisture contact of the protein molded body immediately after molding, but also the subsequent shrinkage due to drying. Is done.

<Protein solution>
The protein solution according to one embodiment contains a protein, a reactive compound, and a solvent in which these are dissolved.

  The concentration of the protein in the protein solution is not particularly limited, and may be, for example, 10 to 30% by mass based on the mass of the protein solution.

  The amount of the reactive compound contained in the protein solution is not particularly limited, and is appropriately determined according to the type of the reactive compound and the like. For example, the amount of the reactive compound may be 100 equivalents or more, 200 equivalents or more, or 300 equivalents or more based on 1 equivalent of the protein. Thereby, a more excellent shrink-proof effect can be obtained. The upper limit of the amount of the reactive compound is not particularly limited, but usually about 300 equivalents or less is sufficient. Here, the equivalent of the reactive compound means the ratio of the amount (mol) of the reactive compound to 1 equivalent (1 mol) of the protein.

  The type of the solvent constituting the protein solution is not particularly limited, and is appropriately determined depending on the type of the protein and the like. The solvent may be, for example, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), hexafluoroisopronol (HFIP), formic acid or a combination thereof. These solvents are suitable, for example, for dissolving spider silk fibroin.

  The protein solution may further contain an inorganic salt. The inorganic salt can function as a protein dissolution promoter. Inorganic salts include, for example, alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, and thiocyanates. Specific examples of the inorganic salt include aluminum phosphate, lithium carbonate, aluminum carbonate, aluminum sulfate, aluminum fluoride, ferric acetate, aluminum acetate, zinc hydroxide, magnesium hydroxide, ferrous hydroxide, and manganese hydroxide. , Chromium hydroxide, ferric hydroxide, aluminum hydroxide, nickel chloride, cobalt chloride, zinc chloride, ferrous chloride, manganese chloride, chromium chloride, ferric chloride, aluminum chloride, lithium nitrate, strontium nitrate, nitric acid Nickel, calcium nitrate, cobalt nitrate, zinc nitrate, magnesium nitrate, ferrous nitrate, manganese nitrate, chromium nitrate, ferric nitrate, aluminum nitrate, lithium bromide, barium bromide, strontium bromide, nickel bromide, odor Calcium bromide, cobalt bromide, zinc bromide, magnesium bromide, ferrous bromide, Manganese chromium, chromium bromide, ferric bromide, aluminum bromide, barium chlorate, strontium chlorate, nickel chlorate, calcium chlorate, cobalt chlorate, zinc chlorate, magnesium chlorate, ferrous chlorate , Manganese chlorate, chromium chlorate, ferric chlorate, aluminum chlorate, rubidium iodide, sodium iodide, copper iodide, lithium iodide, barium iodide, strontium iodide, nickel iodide, calcium iodide , Cobalt iodide, zinc iodide, magnesium iodide, ferrous iodide, manganese iodide, chromium iodide, ferric iodide, aluminum iodide, sodium perchlorate, lead perchlorate, perchloric acid Copper, lithium perchlorate, barium perchlorate, strontium perchlorate, nickel perchlorate, calcium perchlorate, coco perchlorate , Zinc perchlorate, magnesium perchlorate, ferrous perchlorate, manganese perchlorate, chromium perchlorate, ferric perchlorate, aluminum perchlorate, potassium thiocyanate, sodium thiocyanate, thiocyanate Lead acid, copper thiocyanate, lithium thiocyanate, barium thiocyanate, strontium thiocyanate, nickel thiocyanate, calcium thiocyanate, cobalt thiocyanate, zinc thiocyanate, magnesium thiocyanate, ferrous thiocyanate, manganese thiocyanate, thiocyanate Chromic acid, ferric thiocyanate, aluminum thiocyanate, ammonium cyanate, cesium cyanate, rubidium cyanate, potassium cyanate, sodium cyanate, lead cyanate, copper cyanate, lithium cyanate, barium cyanate, cyanide acid Strontium, nickel cyanate, calcium cyanate, cobalt cyanate, zinc cyanate, magnesium cyanate, ferrous cyanate, manganese cyanate, chromium cyanate, ferric cyanate, and aluminum cyanate. At least one of these inorganic salts may be added to the solvent.

  The amount of the inorganic salt contained in the protein solution is not particularly limited, and is appropriately determined according to the type of the inorganic salt, the amount of the protein, and the like. The amount of the inorganic salt is, for example, 1.0 part by mass or more, 5.0 parts by mass or more, 9.0 parts by mass, 15 parts by mass or more, and 20 parts by mass or more based on 100 parts by mass of the total amount of the protein. You may. The amount of the inorganic salt may be, for example, 40 parts by mass or less, 35 parts by mass or less, or 30 parts by mass or less based on 100 parts by mass of the total amount of the protein.

  The protein solution is prepared by a method including dissolving the protein, the reactive compound, and other components as necessary in a solvent. The protein solution may be stirred or shaken for a certain period of time so that the reaction between the first functional group of the reactive compound and the protein proceeds in the protein solution. At that time, the protein solution may be heated if necessary. For example, the protein solution may be heated to 50 ° C. or higher, 90 ° C. or higher, or 120 ° C. or higher. The upper limit of the heating temperature is not particularly limited, but usually 130 ° C. or lower, or about 85 ° C. is sufficient.

<Protein molded body>
The protein molded article produced by the method according to one embodiment is not particularly limited as long as it is obtained by molding using a protein solution, and may be, for example, a fiber (protein fiber) or a film (protein film). . The protein fiber can be obtained by, for example, wet spinning, dry spinning, or dry-wet spinning using a protein solution as a spinning solution. The protein film can be obtained, for example, by casting using a protein solution as a casting solution.

  The molded protein obtained by molding may be stretched before the post-treatment. When producing a protein fiber, the protein fiber formed by spinning may be continuously drawn without being wound. Strain occurs in the molded body due to stretching, which may cause shrinkage.By subjecting the molded body after stretching to post-processing, it is possible to effectively reduce shrinkage due to the strain caused by stretching. it can. This is considered to be because the crosslinked structure of the protein, which can be generated by the post-treatment, immobilizes the protein molecule in a state including distortion. The stretching ratio is not particularly limited, but may be, for example, 3 to 10 times.

  The molded protein obtained by molding may be dried before the post-treatment. This drying is performed, for example, by heating to a temperature at which the reaction of the second functional group does not substantially proceed. Specifically, the heating temperature for drying may be 60 to 90C. Alternatively, drying and post-treatment may be performed simultaneously and in parallel by heating at a higher temperature.

  The protein molded body obtained by molding is subjected to a post-treatment for binding two or more reactive compounds bound to the protein to each other. It is considered that the reaction of the second functional group proceeds by this post-treatment, and as a result, the protein molecules in the molded article are cross-linked via the reactive compound. The means of the post-treatment is appropriately determined depending on the type of the second functional group and the like. For example, the post-treatment can be a heat treatment, UV irradiation, radiation irradiation, addition of a crosslinking accelerator, or a combination thereof. For example, the radically polymerizable unsaturated group as the second functional group can react particularly efficiently by heat treatment, UV irradiation, or radiation irradiation as a post-treatment.

  The heat treatment as the post-treatment may be a dry heat treatment. According to the dry heat treatment, shrinkage of the molded body due to contact with moisture can be more advantageously avoided. The heating temperature and the heating time for the heating treatment are not particularly limited. For example, the heating temperature may be a temperature exceeding 140 ° C., and the heating time may be 30 seconds or more. Thereby, the shrinkage-preventing effect of the finally obtained molded article after the post-treatment can be further enhanced. From a similar viewpoint, the heating temperature for the post-treatment may be 200 ° C. or more, or 240 ° C. or more, and the heating time for the post-treatment may be 30 seconds or more, or 120 seconds or more. The upper limit of the heating temperature is not particularly limited, but may be, for example, 280 ° C. or lower. The upper limit of the heating time is not particularly limited, but may be, for example, 130 seconds or less.

  An example of a method for producing a protein fiber as a protein molded article through a heat treatment as a post-treatment will be described below.

  FIG. 3 is a schematic view showing an example of a spinning device for producing a protein fiber. The spinning device 10 shown in FIG. 3 is an example of a spinning device for dry-wet spinning, and includes an extruder 1, a coagulation device 2 having a coagulation bath 20, a cleaning device 3 having a cleaning bath 21, and a heating device 17. And a drying device 4 in order from the upstream side.

  The extruder 1 has a storage tank 7 in which the stock spinning solution 6 is stored. As the spinning solution 6, the protein solution according to the above-described embodiment is used. The coagulation liquid 11 (for example, methanol) is stored in the coagulation bath 20. The stock spinning solution 6 is pushed out from a nozzle 9 provided with an air gap 19 between the coagulating solution 11 and a coagulating solution 11 by a gear pump 8 attached to the lower end of the storage tank 7. The extruded spinning solution 6 is supplied into the coagulation solution 11 via the air gap 19. The solvent is removed from the spinning dope 6 in the coagulating liquid 11 to coagulate the protein and form protein fibers. The formed protein fibers are guided to the washing bath 21 via the thread guides 18a, 18b, 18c and 18d, and are washed by the washing liquid 12 in the washing bath 21. The washed protein fibers are sent by the first nip roller 13 and the second nip roller 14 installed in the washing bath 21 and introduced into the heating device 17 through the yarn guides 18e, 18f and 18g. At this time, for example, if the rotation speed of the second nip roller 14 is set to be higher than the rotation speed of the first nip roller 13, the protein fiber 36 as a molded article drawn at a magnification corresponding to the rotation speed ratio is obtained. The protein fibers 36 drawn in the washing liquid 12 are dried when they pass through the path 22 in the heating device 17 after leaving the washing bath 21, and are then wound up by the winder 23. In this way, the protein fiber 36 is finally obtained by the spinning device 10 as the wound material 5 wound around the winder 23.

  The coagulation solution may be any solution that can remove the spinning solution from the solvent. Examples of the coagulating liquid include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulation liquid may include water. The temperature of the coagulating liquid may be 0 to 30C. The length of the coagulation liquid tank may be any length as long as the solvent can be efficiently removed, and is, for example, 200 to 500 mm. The residence time of the protein fiber formed by the coagulation in the coagulation liquid may be, for example, 0.01 to 3 minutes, or may be 0.05 to 0.15 minutes. The protein fibers may be drawn (or pre-drawn) in a coagulating liquid. A pre-drawn yarn is formed by pre-drawing.

  The drawing of the protein fibers is performed while heating the washing liquid in the washing bath 21. The cleaning liquid may be, for example, water or a mixed solvent of water and an organic solvent. Stretching performed in a heated washing solution (or solvent) is sometimes referred to as wet heat stretching by those skilled in the art. The temperature of the wet heat stretching (the temperature of the cleaning liquid) may be, for example, 50 to 90 ° C or 75 to 85 ° C. In the wet heat drawing, the undrawn yarn (or pre-drawn yarn) may be drawn, for example, 1 to 10 times, or 2 to 8 times.

  The lower limit of the final draw ratio of the protein fiber is 1 ×, 2 ×, 3 ×, 4 ×, 5 ×, 6 ×, 7 ×, 8 ×, with respect to the undrawn yarn (or pre-drawn yarn). Alternatively, it may be any one of 9 times. The upper limit of the final draw ratio of the protein fiber may be any of 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. .

  FIG. 4 is a schematic diagram showing an example of a heating device for subjecting the protein fibers obtained as described above to a heat treatment as a post-treatment. The heating device 62 shown in FIG. 4 includes the feed roller 42 and the winder 44, and a dry heat plate 64 provided therebetween. The dry heat plate 64 has a dry heat surface 66 extending in a direction from the feed roller 42 to the winder 44.

  The protein fibers 36 are continuously fed from the feed roller 42, and the fed protein fibers 36 are heated while moving along the dry heat surface 66. The conditions of the heat treatment are set so that the reaction of the second functional group of the reactive compound proceeds within the protein fiber 36. As a result, a post-processed protein fiber 38 containing the crosslinked protein is formed. By combining the spinning device 10 of FIG. 3 and the heating device 62 of FIG. 4, it is also possible to continuously produce post-processed protein fibers from the protein solution. In this case, the protein fiber after spinning may be directly subjected to a heat treatment without being wound around a winder, and the protein fiber 36 after spinning may be directly subjected to post-treatment by heating without drying. Is also good.

  The method for producing the protein molded article may further include, after the post-treatment step, a step of immersing the protein fiber in water and drying the immersed protein fiber. Thereby, a molded article having higher dimensional stability can be manufactured.

1. Production of spider silk protein (spider silk fibroin: PRT799) (synthesis of gene encoding spider silk protein and construction of expression vector)
A modified fibroin having an amino acid sequence represented by SEQ ID NO: 13 (hereinafter referred to as “PRT799”) based on the nucleotide sequence and amino acid sequence of fibroin (GenBank Accession No .: P468804.1, GI: 1174415) derived from Nephila clavipes. ").

  The amino acid sequence represented by SEQ ID NO: 13 is obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clabipes, and the N-terminal thereof. And an amino acid sequence represented by SEQ ID NO: 5 (tag sequence and hinge sequence).

  A nucleic acid encoding the designed PRT799 was 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.

Escherichia coli BLR (DE3) was transformed with the obtained pET22b (+) expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 2) so that the OD ₆₀₀ became 0.005. The temperature of the culture was maintained at 30 ° C., and the flask was cultured for about 15 hours until the OD ₆₀₀ reached 5, to obtain a seed culture.

The seed culture solution was added to a jar fermenter to which 500 ml of a production medium (Table 3) had been added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the transformed Escherichia coli was cultured at a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.

  Immediately after the 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 solution was maintained at 37 ° C., and the transformed Escherichia coli was cultured at a constant pH of 6.9. 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 PRT799 expression. 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 expression of PRT799 was confirmed by the appearance of a band having a size corresponding to PRT799 depending on the addition of IPTG.

(Purification of spider silk fibroin)
Two hours after the addition of IPTG, the collected 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 was suspended in 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. The suspension was stirred with a stirrer at 60 ° C. for 30 minutes to dissolve the precipitate. 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 (PRT799) obtained after dialysis was recovered by centrifugation. The water was removed from the collected aggregated proteins using a freeze dryer to obtain a freeze-dried powder of PRT799.

2. Production example 1 of protein fiber
<Preparation of protein solution>
LiCl was dissolved in dimethyl sulfoxide (DMSO) as a solvent to a concentration of 4.0% by mass. A dry powder of spider silk fibroin (PRT799) was added to the obtained solution so that the concentration became 24% by mass. Subsequently, 1,1- (bisacryloyloxymethyl) ethyl isocyanate (BEI) as a reactive compound was newly added, and the mixture was stirred with a shaker so as to be uniform. The amount of BEI was 300 equivalents (300 mol) per equivalent (1 mol) of spider silk fibroin (PRT799). The resulting mixture was stirred for 5 hours using a mechanical stirrer to dissolve the protein in the solvent and to react the protein with BEI. Thereafter, insolubles and bubbles were removed to obtain a protein solution. The viscosity of the protein solution was 11000 cP (centipoise) at 90 ° C.

<Spinning>
The obtained protein solution was used as a spinning dope, and spun and drawn protein fibers were formed by dry-wet spinning using the spinning apparatus 10 shown in FIG. The formed protein fibers were dried and wound up. The conditions of the dry-wet spinning are as follows.
Extrusion nozzle diameter: 0.2mm
Temperature of coagulation liquid (methanol): 5 ° C
Water washing bath stretch ratio: 5 times Drying temperature: 60 ° C

(Dry heat treatment)
The obtained protein fiber was subjected to dry heat treatment as a post-treatment using the heating device 62 shown in FIG. 4, and the protein fiber after the dry heat treatment was wound up. The conditions of the dry heat treatment are as follows.
Delivery speed: 25 cm / min
Winding speed: 25cm / min
Dry heat plate length: 50cm
Dry hot plate temperature: 240 ° C

Comparative Example 1
The protein fiber after spinning and drawing was evaluated as the protein fiber of Comparative Example 1 without being subjected to the dry heat treatment.

Comparative Example 2
A protein solution was prepared in the same manner as in Example 1 except that BEI was not added to the protein solution. Using the obtained protein solution as a spinning dope, protein fibers were obtained by spinning and stretching in the same manner as in Example 1. This was evaluated as the protein fiber of Comparative Example 2 without being subjected to the dry heat treatment.

3. Evaluation of shrinkage For each protein fiber of Examples and Comparative Examples, shrinkage due to contact with moisture immediately after production (sometimes referred to as “primary shrinkage”) and subsequent shrinkage with drying (“secondary shrinkage” are referred to as “secondary shrinkage”). A) was evaluated.

<Primary shrinkage>
A plurality of test protein fibers each having a length of 30 cm were cut out from the wound protein fibers of Examples and Comparative Examples. By bundling the plurality of protein fibers, a protein fiber bundle having a fineness of 150 denier was obtained. A 0.8 g lead weight was attached to each protein fiber bundle, and in this state, each protein fiber bundle was immersed in water at room temperature for 15 hours or more. Thereafter, the length of each protein fiber bundle was measured in water. The length of the protein fiber in water was measured with a 0.8 g lead weight attached to the protein fiber in order to eliminate the shrinkage of the protein fiber in hot water. Next, the shrinkage rate (%, primary shrinkage rate) when each protein fiber bundle was immersed in water was calculated according to the following formula I. In Formula I, L0 indicates the initial length of the protein fiber bundle before immersion (here, 30 cm), and Lw1 indicates the length of the protein fiber bundle after immersion. Primary shrinkage corresponds to the change in protein fiber length following initial contact with moisture after production.
Primary shrinkage = {(L0−Lw1) / Lw1} × 100 (formula I)

<Secondary shrinkage>
After the first dipping for evaluation of primary shrinkage, the protein fiber bundle was removed from the water. The removed protein fiber bundle was dried by heating at room temperature for 5 hours or more with a 0.8 g lead weight attached (first drying). After drying, the length of each protein fiber bundle was measured. Thereafter, each protein fiber bundle was immersed again in water at room temperature for 15 hours or more, and the length of each protein fiber bundle was measured in water. These immersion and drying operations were repeated until immersion was performed three times and drying was performed three times. Then, the shrinkage rate (%, secondary shrinkage rate) of secondary shrinkage due to drying of the protein fiber bundle after immersion in water was calculated according to the following formulas II, III and IV. In these formulas, n indicates the number of times of immersion or drying, Lwn indicates the length of the protein fiber bundle after the n-th immersion, and Ldn indicates the length of the protein fiber bundle after the n-th drying. Here, n is 3. The secondary shrinkage corresponds to the reversible change in length of the protein fiber after production, after contraction by contact with moisture, and after drying.
Lwet = (Lw1 + Lw2 +... + Lwn) / n (Formula II)
Ldry = (Ld1 + Ld2 +... + Ldn) / n (Formula III)
Secondary shrinkage = {(Lwet-Ldry) / Lwet} × 100 (Formula IV)

  As shown in Table 4, the protein fibers of Example 1 had a primary shrinkage rate of 8.1% when immersed in water and a secondary shrinkage rate of 7% upon drying after immersion. It was low. When the appearance of the protein fiber of Example 1 after immersion was visually checked, no shrinkage was observed. In contrast, the protein fibers of Comparative Examples 1 and 2 showed large values for both the primary shrinkage and the secondary shrinkage.

  From these experimental results, it can be seen that the protein fibers produced by the method according to the present invention are able to extremely effectively suppress the shrinkage due to the initial contact with moisture after production and the subsequent shrinkage due to drying. Was confirmed.

Change in solubility of fiber by heat treatment A very small amount of the protein fiber after heat treatment was added to 2 mL of dimethyl sulfoxide (DMSO) solution containing LiCl at a concentration of 4.0% by mass. After heating the DMSO solution at 90 ° C. for 24 hours, the state of the contents was confirmed. Spider silk fibroin (PRT799) was originally soluble in DMSO, but the protein fibers did not dissolve and the fiber shape was maintained. Was left. This is considered to be because the second functional groups reacted with each other by the heat treatment, and the crosslinking of the protein progressed. As a result, the solubility of the protein fiber was reduced.

4. Confirmation of reaction between spider silk fibroin and BEI 4-1. Production of spider silk protein (spider silk fibroin: PRT410) Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P468804.1, GI: 1174415) derived from Nephila clavipes, it is represented by SEQ ID NO: 9. (Hereinafter, also referred to as “PRT410”) having a modified amino acid sequence. A lyophilized powder of PRT410 was obtained in the same manner as in PRT799.

4-2. Treatment of spider silk protein with BEI solution Lyophilized powder of PRT410 was added to DMSO containing 5.0% by mass of LiCl to a concentration of 24% by mass. Thereto was added BEI as a reactive compound. The amount of BEI was 100 equivalents (100 mol) per 1 equivalent (1 mol) of PRT410. The resulting mixture was stirred for 5 hours using a magnetic stirrer to dissolve LiCl and BEI in DMSO. Thereafter, ethanol was added to precipitate a white precipitate. The precipitate was washed three times in water while crushing, and then freeze-dried. The resulting white lyophilized powder was analyzed by SDS-PAGE. As a result, it was confirmed that a new band appeared slightly above the band corresponding to PRT410. This band is considered to be a component derived from the reaction between PRT410 and BEI.

4-3. Analysis by NMR Lyophilized powder of spider silk fibroin treated with the BEI solution by the above method was dissolved in heavy DMSO to obtain a sample for NMR measurement. ¹ H NMR of the obtained sample was measured by a nuclear magnetic resonance apparatus (400 MHz, manufactured by JEOL). FIG. 5 is a ¹ H NMR spectrum of spider silk fibroin after treatment with a BEI solution. The ¹ H NMR spectrum of FIG. 5 was compared with the ¹ H NMR spectrum obtained using untreated lyophilized powder of PRT410 or a heavy DMSO solution of BEI. As a result, after the treatment with the BEI solution, it was confirmed that a characteristic signal derived from BEI appeared around 5.89 to 6.42 ppm in addition to a broad signal derived from spider silk fibroin (PRT410). . From this, it was confirmed that the measurement sample contained both spider silk fibroin and BEI.

4-4. DOSY
The sample prepared in “4-3. Analysis by NMR” was analyzed by ¹ H NMR by the DOSY method. FIG. 6 is a DOSY method ¹ H NMR spectrum of spider silk fibroin treated with the BEI solution. In FIG. 6, (a) shows a spectrum without application of a magnetic field gradient, and (b) shows a spectrum with application of a magnetic field gradient. FIG. 7 is an enlarged view showing a region B in FIG. A magnetic field gradient was applied until the signals of water (3.35 ppm) and DMSO (2.49 ppm) observed in region A in the figure disappeared. In this state, the decay behavior of the BEI signal in region B was comparable to the PRT410 signal. If BEI did not react with PRT410 and existed in a low molecular weight state, the signal should exhibit a decay behavior close to that of the low molecular weight components water and DMSO, and should disappear. Therefore, the decay behavior of the BEI signal attenuated to the same extent as the PRT410 signal strongly suggests that BEI is reacted with and bound to the high molecular weight PRT410.

  DESCRIPTION OF SYMBOLS 1 ... Extrusion apparatus, 2 ... Solidification apparatus, 3 ... Cleaning apparatus, 4 ... Drying apparatus, 6 ... Spinning stock solution, 10 ... Spinning apparatus, 12 ... Cleaning liquid, 13 ... First nip roller, 14 ... Second nip roller, 19 ... Air gap , 20: coagulation bath, 64: dry heat plate, 36: protein fiber, 42: feed roller, 44: winder, 62: heating device.

Claims (20)

A protein solution containing a protein, a reactive compound, and a solvent in which the protein and the reactive compound are dissolved, wherein the reactive compound has a first functional group and a second functional group different from the first functional group. A compound wherein the first functional group is a group capable of binding the protein and the reactive compound by reaction with the protein, and wherein the second functional group is a reaction of two or more second functional groups. Preparing a protein solution, which is a group capable of binding the two or more reactive compounds to each other,
A step of obtaining a molded body containing the protein by molding using the protein solution as a stock solution,
Subjecting the molded body to a post-treatment for bonding two or more of the reactive compounds to each other by a reaction of two or more of the second functional groups;
A method for producing a protein molded article, comprising:   The method according to claim 1, further comprising a step of stretching the molded body before the step of subjecting the molded body to the post-treatment.   The method according to claim 1, wherein the molded body is a protein fiber.   The method according to any one of claims 1 to 3, wherein the protein comprises a structural protein.   The method according to claim 4, wherein the structural protein is spider silk fibroin.   The method according to any one of claims 1 to 5, wherein the first functional group is an isocyanate group, and the second functional group is a radically polymerizable unsaturated group.   The method according to any one of claims 1 to 6, wherein the post-treatment is a heat treatment of the molded body.   The method of claim 7, wherein the heat treatment is heating the compact to a temperature greater than 140C.   The method according to claim 7, wherein the time during which the molded body is subjected to the heat treatment is 30 seconds or more.   The method according to any one of claims 1 to 9, wherein the amount of the reactive compound in the protein solution is 100 equivalents or more based on 1 equivalent of the protein. A protein molded article containing a protein cross-linked by a reactive compound,
The reactive compound is a compound having a first functional group and a second functional group different from the first functional group, and the protein reacts with the protein by reacting the first functional group with the protein. A protein molded article, wherein a compound is bound to a protein, and two or more of the reactive compounds are bound to each other by a reaction of two or more of the second functional groups, whereby the protein is cross-linked.   The protein molded product according to claim 11, which is a protein fiber.   The protein molded product according to claim 11, wherein the protein includes a structural protein.   14. The protein molded article according to claim 13, wherein the structural protein is spider silk fibroin.   The protein molded article according to any one of claims 11 to 14, wherein the first functional group is an isocyanate group, and the second functional group is a radically polymerizable unsaturated group. A protein solution containing a protein and a reactive compound and a solvent in which these are dissolved,
The reactive compound is a compound having a first functional group and a second functional group different from the first functional group, and the first functional group reacts with the protein by reacting with the protein. A protein solution, which is a group capable of binding to a compound, wherein the second functional group is a group capable of binding two or more reactive compounds to each other by a reaction of two or more second functional groups.   The protein solution according to claim 16, which is a spinning solution for spinning protein fibers.   The protein solution according to claim 16 or 17, wherein the protein comprises a structural protein.   19. The protein solution according to claim 18, wherein the structural protein is spider silk fibroin.   The protein solution according to any one of claims 16 to 19, wherein the first functional group is an isocyanate group, and the second functional group is radically polymerizable unsaturated.