JP2020054487A - Acid release body - Google Patents

Abstract

To provide an acid release body that holds an acid release ability without having a complicated structure, and can exhibit an acid release ability as appropriate.SOLUTION: An acid release body has a protein composition containing esterified protein, and an acid or a base.SELECTED DRAWING: None

Description

  The present invention relates to an acid emitter.

  Acids have a bactericidal effect, a deodorant effect, a deodorant effect, etc., and are therefore used as various germicides, deodorants and the like. For example, Patent Literature 1 discloses an insole as an acid releaser, which has an effect of preventing bacterial growth and malodor by releasing acetic acid. Further, for example, Japanese Patent Application Laid-Open No. H11-163873 is characterized by comprising a sheath made of a fiber-forming polymer and a core made of a low-melting polymer containing a porous substance holding a sustained-release component. Release-in-sheath composite fibers are disclosed.

JP-A-63-209604 JP-A-6-322612

  However, the acid releasing body as disclosed in Patent Document 1 is formed by using a porous hollow fiber impregnated with an acid, and is configured such that the acid is released to the outside through a through hole of the hollow fiber. Therefore, it is difficult to control the release of acid, and it is difficult to maintain the ability to release acid such as a volatile acid for a long period of time. Then, in order to maintain the acid releasing ability for a long period of time, as disclosed in Patent Document 2, it is conceivable to make the structure of the acid releasing body a complex structure such as a double structure. There is a problem in that the production of the acid-releasing body having the above-mentioned problem is troublesome.

  Therefore, an object of the present invention is to provide an acid releasing body that can maintain an acid releasing ability without having a complicated structure and can exert an acid releasing ability as needed.

  One aspect of the present invention relates to an acid emitter comprising a protein composition comprising an esterified protein and an acid or a base.

  In the acid releaser according to the present invention, the acid or base contained in the acid releaser functions as a catalyst for a hydrolysis reaction of the esterified protein, and releases an acid (carboxylic acid) by hydrolysis of the ester group. . For this reason, the acid releasing body according to the present invention can exhibit an acid releasing ability by contact with water. Since the acid releasing body according to the present invention releases an acid based on such an action mechanism, the acid releasing ability is maintained even if it does not have a complicated structure, and if necessary (contact with moisture) ) Can exert an acid releasing ability.

  In one aspect, the protein may be a structural protein.

  In one aspect, the structural protein may be a modified fibroin.

  In one embodiment, the modified fibroin may be a modified spider silk fibroin.

  In one embodiment, the protein composition may be at least one selected from the group consisting of a fiber, a film, a gel, a porous body, and a molded article.

  In one embodiment, the esterified protein may include at least one selected from the group consisting of formate, acetate, and propionate.

  ADVANTAGE OF THE INVENTION According to this invention, the acid release ability is maintained, without providing a complicated structure, and the acid release body which can exhibit an acid release ability as needed is provided.

It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. It is a figure which shows the distribution of the value of z / w (%) of the fibroin derived from nature. It is a figure which shows the distribution of the value of x / y (%) of fibroin derived from nature. 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 graph which plotted the absorbance ratio (P1 / P2) which reflected the residual amount of the ester group at the time of contacting the acid releasing body which concerns on one Embodiment with moisture with respect to the number of contact days. P1: Peak height of 1725 cm ⁻¹ (peak based on C ＝ O of ester). P2: peak height at 1445 cm ^-1 (peak based on amide III of the protein).

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

(Acid emitter)
The acid releaser according to the present embodiment contains a protein composition containing an esterified protein, and an acid or a base.

  In this embodiment, the protein composition comprises an esterified protein. In the present specification, “esterified protein” means a protein containing an ester group formed by an ester bond between a hydroxyl group of the protein and a carboxylic acid. The esterified protein may include at least one selected from the group consisting of formate, acetate, and propionate.

(protein)
Examples of the protein according to the present embodiment (hereinafter, sometimes referred to as “target protein”) include a natural protein and a recombinant protein (artificial protein). In addition, examples of the recombinant protein include any protein that can be produced on an industrial scale, such as a protein that can be used for industrial purposes, a protein that can be used for medical purposes, and a structural protein. Specific examples of proteins that can be used for industrial or medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membranes or transport proteins, antigens used for vaccination, vaccines, antigen-binding proteins, immunostimulating proteins, Mention may be made of allergens, full length antibodies or antibody fragments or derivatives. Specific examples of the structural protein include spider silk (spider silk), silkworm silk, keratin, collagen, elastin, and resilin, and proteins derived therefrom. Further, the structural protein may be, for example, a modified fibroin described later, and a modified spider silk fibroin is preferable.

As a protein derived from spider silk or silkworm silk which is a fibroin-like protein, for example, formula 1: [(A) _n motif-REP1] _m or formula 2: [(A) _n motif-REP1] _m- (A) _n Examples include proteins containing a domain sequence represented by a motif. The fibroin may have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.

As used herein, the term “modified fibroin” means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be the same as the amino acid sequence of naturally occurring fibroin. As used herein, “naturally-derived fibroin 1” is also represented by Formula 1: [(A) _n motif-REP1] _m or Formula 2: [(A) _n motif-REP1]-(A) _n motif. Is a protein containing the domain sequence to be determined.

  "Modified fibroin" may be a directly used amino acid sequence of a naturally occurring fibroin, or a modified amino acid sequence based on the amino acid sequence of a naturally occurring fibroin (for example, cloned naturally occurring fibroin). The amino acid sequence may be modified by modifying the gene sequence of fibroin), or may be artificially designed and synthesized without using naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence may be used). Which have a desired amino acid sequence by chemical synthesis).

As used herein, the term “domain sequence” refers to a crystalline region unique to fibroin (typically, corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence). The amino acid sequence represented by Formula 1: [(A) _n motif-REP1] m or Formula 2: [(A) _n motif-REP1] m- (A) _n motif Means an array. Here, the (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (A) The _number of amino acid residues of the _n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. . (A) The ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Specific examples of the modified fibroin include a modified fibroin (first modified fibroin) derived from a large spinal cord marker protein produced in a spider's large ampullate gland, and a domain sequence having a reduced content of glycine residues. (A second modified fibroin), (A) a modified fibroin having a domain sequence with a reduced content of the _n motif (a third modified fibroin), a glycine residue content, and (A) _n Modified fibroin having a reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence containing a region having a large hydrophobicity index (fifth modified fibroin), and glutamine residue content Modified fibroin having a reduced domain sequence (sixth modified fibroin).

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

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

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

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

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

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

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

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

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

The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the (A) _n motif located at the most C-terminal side from the above domain sequence, to the above domain sequence The total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus is represented by z, From, when the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is defined as w, z / w is 30% or more; It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).

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

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

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

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

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

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

  As more specific examples of the second modified fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met -PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, Modified fibroin can be mentioned.

The modified fibroin of (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. The amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In this figure, one [(A) _n motif-REP] is inserted. The amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) _n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some amino acids on the C-terminal side have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7. The amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). Is repeated four times with a predetermined hinge sequence and His tag sequence added to the C-terminal.

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

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

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

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

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

  Examples of the tag sequence include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including a His tag sequence and a hinge sequence).

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

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

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

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

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

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

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

  The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

When the third modified fibroin is a modified fibroin in which at least seven of the (A) _n motifs present in a plurality in the domain sequence are composed of only alanine residues, x / y should be 46.4% or more. Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferred that there is. The upper limit of x / y is not particularly limited, and may be 100% or less.

Here, x / y in naturally occurring fibroin will be described. First, as described above, when fibroin whose amino acid sequence information was registered in the NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Among all the extracted fibroins, x / y was calculated from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by Formula 1: [(A) _n motif-REP] _m by the above-described calculation method. Was calculated. FIG. 3 shows the results when the knurl ratio is 1: 1.9 to 4.1.

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

The third modified fibroin, for example, deletes one or more of the sequence encoding the (A) _n motif from the cloned natural fibroin gene sequence such that x / y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed so that x / y is 64.2% or more based on the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. In each case, in addition to the modification corresponding to the deletion of 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. Amino acid sequence modification corresponding to the above may be performed.

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

The modified fibroin of (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 17 differs from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin in that every two amino acids from the N-terminal side to the C-terminal side (A) _n The motif was deleted, and one [(A) _n motif-REP] was inserted before the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.

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

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

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

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

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

  As more specific examples of the modified fibroin containing a tag sequence, (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) Modified fibroin comprising a sequence or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .

  The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding SEQ ID NO: 11 to the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. (Including a His tag sequence and a hinge sequence).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

For example, when “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” are extracted at 20 locations (without duplication), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2 The region of .6 or more would contain 20 consecutive 4 amino acid residues (no duplication), and p would be 20 × 4 = 80. For example, when two “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of four consecutive amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “−1” is a deduction for duplication). For example, in the case of the domain sequence shown in FIG. 4, there are seven consecutive “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without overlapping. 28. Also, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (the last (A) _n motif on the C-terminal side is not included). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

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

  The fifth modified fibroin is, for example, one or a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the cloned natural fibroin is satisfied so as to satisfy the above-mentioned p / q conditions. Substituting a negative amino acid residue with a hydrophobic amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can also be obtained by designing an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP have been replaced by amino acid residues with a higher hydrophobicity index and / or one or more amino acid residues in the REP as compared to naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, a modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues may be performed. .

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

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

  The modified fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 19 consists of three amino acid residues every other REP, except for the domain sequence of the terminal at the C-terminal side, with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410). In this amino acid sequence, two amino acid sequences (VLI) were inserted, some glutamine (Q) residues were further substituted with serine (S) residues, and some C-terminal amino acids were deleted. The amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). is there. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8.

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

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

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

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

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

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

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

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

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

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

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

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

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

In the present specification, the “GPGXX motif content” is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: fibroin containing a domain sequence represented by [(A) _n motif-REP] _m- (A) _n motif (modified fibroin or naturally occurring fibroin) Fibroin), the number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total number by 3 (ie, the total number of G and P in the GPGXX motif) is represented by s, and the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is represented by (A) When the total number of amino acid residues of all REPs excluding the (A) _n motif is represented by t, the content of the GPGXX motif is calculated as s / t. It is.

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

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

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

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

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

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

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

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

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

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

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

  As more specific examples of the sixth modified fibroin, (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met) -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or sequence No. 42 (Met-PRT1028), or a modified fibroin comprising the amino acid sequence represented by (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It can be mentioned modified fibroin comprising an amino acid sequence having a sequence at least 90% sequence identity.

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

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

  The amino acid sequence represented by SEQ ID NO: 32 is obtained by substituting all VFs for QQ in a sequence obtained by repeating twice the domain of the 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410), In addition, the remaining Q is replaced with I.

  The amino acid sequence represented by SEQ ID NO: 41 (Met-PRT917) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with LI and substituting the remaining Q with V. The amino acid sequence represented by SEQ ID NO: 42 (Met-PRT1028) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with IF, and substituting the remaining Q with T.

  The amino acid sequences represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 all have glutamine residues. The group content is 9% or less (Table 2).

  The modified fibroin of (6-i) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It may consist of the amino acid sequence shown.

The modified fibroin of (6-ii) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown. The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. A protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

  As more specific examples of the modified fibroin containing a tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 44 (PRT1028), or ( 6-iv) The amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 and 90 % Modified amino acids comprising an amino acid sequence having at least 50% sequence identity It can be mentioned.

  The amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 have SEQ ID NO: 25, respectively. SEQ ID NO: 11 at the N-terminal of the amino acid sequence represented by SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: Amino acid sequence (including a His tag sequence and a hinge sequence). Since only a tag sequence was added to the N-terminus, there was no change in the glutamine residue content, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

  The modified fibroin of (6-iii) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It may consist of the amino acid sequence shown.

The modified fibroin of (6-iv) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. A protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

  The modified fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. Modified fibroin having both of the following characteristics may be used.

As spider silk and a protein derived from the weft protein, for example, a protein containing a domain sequence represented by Formula 3: [REP2] _o (wherein, in Formula 3, REP2 is Gly-Pro-Gly-Gly- X represents an amino acid sequence composed of X, wherein X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr), and valine (Val), and o is an integer of 8 to 300. Is shown.) Spider silk contains many structures having hydroxyl groups such as serine and tyrosine, and these hydroxyl groups are listed as functional groups that can be converted into esters.

  Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 47 can be mentioned. The amino acid sequence represented by SEQ ID NO: 47 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 linked, and the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminus of the linked sequence; is there.

As a collagen-derived protein, for example, a protein containing a domain sequence represented by Formula 4: [REP3] _p (where _p represents an integer of 5 to 300. In Formula 4, REP3 is Gly-X-Y Wherein 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.) . Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 48. The amino acid sequence represented by SEQ ID NO: 48 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: 11 (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 (where, in Formula 5, q represents an integer of 4 to 300. REP4 is Ser-J-J1- 1 shows an amino acid sequence composed of Tyr-Gly-U-Pro, wherein J represents any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. And preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser .. A plurality of REP4s may have the same amino acid sequence or different amino acid sequences. ). Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 49. In the amino acid sequence represented by SEQ ID NO: 49, in the amino acid sequence of resilin (GenBank Accession No. NP 611157 of NCBI, Gl: 246654243), Thr at the 87th residue is replaced with Ser, and Asn at the 95th residue is substituted. In which the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced by Asp.

  Examples of elastin-derived 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 include a protein containing the amino acid sequence represented by SEQ ID NO: 50. The amino acid sequence represented by SEQ ID NO: 50 corresponds to the amino acid sequence represented by SEQ ID NO: 11 (tag sequence) at the N-terminus of the amino acid sequence from residues 121 to 390 of the amino acid sequence of NCBI GenBank accession number AAC98395. And a hinge sequence).

  Examples of keratin-derived proteins include, for example, Capra hircus type I keratin and the like. Specific examples include a protein comprising the amino acid sequence represented by SEQ ID NO: 51 (the amino acid sequence of GenBank Accession No. ACY30466 of NCBI).

(Protein production method)
A protein can be expressed, for example, by expressing the nucleic acid with a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced.

  The method for producing the gene encoding the protein is not particularly limited. For example, the gene 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 by chemical synthesis. The method for chemically synthesizing genes 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 the oligonucleotides automatically synthesized by the method such as PCR. At this time, in order to facilitate purification and confirmation of the protein, a gene 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 may be synthesized. Good.

  The regulatory sequence is a sequence that controls the expression of the 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 can induce the expression of a recombinant 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. As the expression vector, those capable of autonomous replication in a host cell or integration into the chromosome of a host and containing a promoter at a position where a nucleic acid encoding a protein can be transcribed are suitably used.

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

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

  When a prokaryote is used as a host, examples of a vector for introducing a nucleic acid encoding a recombinant protein include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, and pUB110. And 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 genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

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

  As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, 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, a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.

  The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolyzate, acetic acid and propionic acid, and the like. Organic acids and alcohols such as ethanol and propanol can be used. As the nitrogen source, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used. As the inorganic salt, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

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

  If necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture. 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 commonly used for isolating and purifying proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, 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). Electrophoretic 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. And other methods alone or in combination. Goods can be obtained.

  When the protein is expressed in an insoluble form in the cell, the host cell is similarly recovered, crushed, and centrifuged to collect the insoluble form of the recombinant protein as a precipitate fraction. The recovered insoluble form of the 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 technique 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.

(Method for producing esterified protein)
The esterified protein according to the present embodiment can be obtained, for example, by reacting a protein produced by the above-described method with a carboxylic acid, so that the protein is contained in a hydroxyl group (for example, a serine residue, a tyrosine residue, or a threonine residue). It can be obtained by forming an ester group by a dehydration condensation reaction between a (hydroxyl group) and a carboxylic acid (esterification step).

  As the carboxylic acid, for example, formic acid, acetic acid, dichloroacetic acid, trifluoroacetic acid, propionic acid, butanoic acid, isobutyric acid, pentanoic acid, caproic acid, caprylic acid, capric acid, monocarboxylic acid such as benzoic acid, capric acid, Saturated aliphatic carboxylic acids such as lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, montanic acid, melissic acid, celloplastic acid, undecylenic acid, oleic acid, elaidic acid, setrein Acids, erucic acid, brassic acid, sorbic acid, linoleic acid, linolenic acid, arachidonic acid, propiolic acid, unsaturated aliphatic carboxylic acids such as stearic acid, and oxalic acid, malonic acid, succinic acid, glutaric acid, adipine Acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, Ndekanin acid, Burashirin acid, maleic acid, fumaric acid, dicarboxylic acids such as glutaconic. The carboxylic acid may be in the form of an acid anhydride or an acid chloride.

  The esterification step can be performed, for example, by dissolving the protein produced by the above-described method in a solvent containing a carboxylic acid.

  The esterification step may be performed at room temperature, or may be maintained at various heating temperatures to dissolve the protein in a solvent containing a carboxylic acid. The holding time of the heating temperature is not particularly limited, but may be 10 minutes or more. In view of industrial production, it is preferably 10 to 120 minutes, more preferably 10 to 60 minutes, and still more preferably 10 to 30 minutes. The holding time at the heating temperature may be appropriately set under the condition that the protein is sufficiently dissolved and impurities (other than the target protein) are less dissolved.

  The amount of the carboxylic acid added to dissolve the protein is not particularly limited as long as the protein can be dissolved. When the purified protein is dissolved, the amount of the carboxylic acid to be added is determined by the ratio of the volume (mL) of the carboxylic acid to the weight (g) of the protein (dry powder containing the protein) (volume (mL) / weight (g)). May be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times. When dissolving the protein in the host cell in which the protein was expressed, the amount of the carboxylic acid added was determined by the ratio of the volume (mL) of the carboxylic acid to the weight (g) of the host cell (volume (mL) / weight (g)). May be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times.

  The solvent containing a carboxylic acid may contain an inorganic salt. By adding an inorganic salt to the solvent, it is possible to increase the solubility of the protein.

  Examples of inorganic salts that can be added include alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, thiocyanates, perchlorates, and the like. Examples of the alkali metal halide include potassium bromide, sodium bromide, lithium bromide, potassium chloride, sodium chloride, lithium chloride, sodium fluoride, potassium fluoride, cesium fluoride, potassium iodide, sodium iodide, Lithium iodide and the like can be mentioned. Examples of the alkaline earth metal halide include calcium chloride, magnesium chloride, magnesium bromide, calcium bromide, magnesium iodide, calcium iodide and the like. Examples of the alkaline earth metal nitrate include calcium nitrate, magnesium nitrate, strontium nitrate, barium nitrate and the like. Examples of the thiocyanate include sodium thiocyanate, ammonium thiocyanate, guanidinium thiocyanate and the like. Examples of the perchlorate include ammonium perchlorate, potassium perchlorate, calcium perchlorate, silver perchlorate, sodium perchlorate, magnesium perchlorate and the like. One of these inorganic salts may be used alone, or two or more thereof may be used in combination. Suitable inorganic salts include alkali metal halides and alkaline earth metal halides. Specific examples of suitable inorganic salts include lithium chloride, calcium chloride and the like. The addition amount (content) of the inorganic salt may be 0.5% by mass or more and 10% by mass or less, or 0.5% by mass or more and 5% by mass or less based on the total mass of the carboxylic acid.

(Protein composition)
The protein composition according to the present embodiment only needs to include the above-described esterified protein, and may have any shape (for example, liquid, paste, or solid). In one embodiment, the protein composition may be at least one molded product selected from the group consisting of a fiber, a film, a gel, a porous material, and a molded product. These molded products can be manufactured according to a conventional method.

  The molded article is obtained by, for example, a step of dissolving a protein in a solvent to obtain a protein solution (hereinafter, also referred to as a “dissolution step”), and forming the obtained protein solution into a desired shape (for example, a fiber, a film, a porous body, (Molded body or the like) to obtain a molded body.

[Dissolution step]
The dissolving step is a step of dissolving the protein in a solvent to obtain a protein solution.

  The solvent is not particularly limited as long as it can dissolve the protein, and examples thereof include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), carboxylic acids such as formic acid, and hexafluoroisopropanol (HFIP). No. An inorganic salt may be added to the solvent to increase the solubility of the protein. Examples of the inorganic salt and the amount thereof may be the same as those described in the above embodiment.

  It is preferable that the solvent contains a carboxylic acid such as formic acid. By using a solvent containing a carboxylic acid such as formic acid as a solvent, the esterification step and the dissolution step described above can be combined.

  The amount of the solvent to be added is not particularly limited as long as it can dissolve the protein.

  When dissolving the purified protein, the amount of the solvent added is expressed as a ratio (volume (mL) / weight (g)) of the volume (mL) of the acid to the weight (g) of the protein (dry powder containing the protein). It may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times.

  When dissolving the protein in the host cell in which the protein has been expressed, the amount of the solvent to be added may be 1 to 1 as the ratio of the acid (mL) to the weight (g) of the host cell (volume (mL) / weight (g)). It may be 100 times, may be 1 to 50 times, may be 1 to 25 times, may be 1 to 10 times, and may be 1 to 5 times.

  In the lysis step, a purified protein may be used as a protein to be lysed (hereinafter, also referred to as “target protein”), or a protein in a host cell expressing the protein (recombinant protein) may be used. Good. The purified protein may be a protein purified from a host cell that expressed the protein. When dissolving a protein in a host cell as a target protein, the host cell is brought into contact with an acid to dissolve the protein in the host cell in the acid. The host cell may be one that expresses the target protein, and may be, for example, an intact cell or a cell that has been subjected to treatment such as destruction. Alternatively, cells that have already been subjected to a simple purification treatment may be used.

  The method for purifying the protein from the host cell in which the protein has been expressed is not particularly limited. For example, the methods described in Japanese Patent Nos. 6077570 and 6077569 can be used.

  The dissolving step may be performed at room temperature, or may be maintained at various heating temperatures to dissolve the protein in the solvent. The holding time of the heating temperature is not particularly limited, but may be 10 minutes or more. In view of industrial production, it is preferably 10 to 120 minutes, more preferably 10 to 60 minutes, and still more preferably 10 to 30 minutes. The holding time at the heating temperature may be appropriately set under the condition that the protein is sufficiently dissolved and impurities (other than the target protein) are less dissolved.

  The protein solution may be removed of insolubles as necessary. That is, the method for producing a protein molded article of the present embodiment may include a step of removing insolubles from the protein solution, if necessary, after the dissolving step. Examples of a method for removing insolubles from the protein solution include a general method such as centrifugation, filtration with a drum filter, a press filter, or the like. In the case of filtration using a filter, insoluble matter can be more efficiently removed from the protein solution by using a filter aid such as celite and diatomaceous earth together with a precoating agent.

  The protein solution contains a protein and a solvent in which the protein is dissolved (solvent for dissolution). The protein solution may contain contaminants that were included with the protein in the dissolution step. The protein solution may be a solution for molding a protein molded article.

  The content of the protein in the protein solution may be 5% by mass to 35% by mass, or 5% by mass to 50% by mass based on the total amount of the protein solution.

[Molding process]
The molding step is a step of molding a protein composition as a molded article using a protein solution. The shape of the protein composition as a molded article (hereinafter, also referred to as “protein molded article”) is not particularly limited, and examples thereof include fibers, films, porous bodies, and molded articles.

  It is preferable to adjust the protein concentration and viscosity of the protein solution depending on the protein molded product to be molded.

  The method of adjusting the concentration of the protein in the protein solution is not particularly limited, for example, a method of increasing the protein concentration by evaporating the solvent by distillation, a method of using a protein having a high protein concentration in the dissolution step, or A method of reducing the amount of the solvent to be added to the amount of the protein may be used.

  The viscosity suitable for spinning is generally 10 to 50,000 cP (centipoise), and the viscosity can be measured using, for example, "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd. When the viscosity of the protein solution is not in the range of 10 to 10,000 cP (centipoise), the viscosity of the protein solution may be adjusted to a viscosity that allows spinning. For adjusting the viscosity, the above-described method or the like can be used. The solvent may include any suitable inorganic salts described above.

  When the protein molded product is a protein fiber, the protein content (concentration) in the protein solution may be adjusted to a concentration and a viscosity that allow spinning, if necessary. The method for adjusting the concentration and viscosity of the protein is not particularly limited. Examples of the spinning method include wet spinning. When a protein solution adjusted to a concentration and viscosity suitable for spinning is applied to a coagulation solution as a dope solution, the protein coagulates. At this time, by applying the protein solution as a thread-like liquid to the coagulation liquid, the protein is coagulated into a thread and a thread (undrawn thread) can be formed. The formation of the undrawn yarn can be performed, for example, according to the method described in Japanese Patent No. 5584932.

  Hereinafter, an example of wet spinning will be described, but the spinning method is not particularly limited, and may be dry wet spinning or the like.

Wet spinning-drawing (a) Wet spinning The coagulating liquid may be any solution that can remove the solvent. It is preferable to use a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol or acetone as the coagulating liquid. The coagulating liquid may include water. The temperature of the coagulation liquid is preferably from 5 to 30C from the viewpoint of spinning stability.

  The method of applying the protein solution as a thread-like liquid is not particularly limited, and includes, for example, a method of extruding from a spinneret into a coagulating liquid in a desolvation tank. An undrawn yarn is obtained by coagulation of the protein. The extrusion speed when extruding the protein solution into the coagulation liquid can be appropriately set according to the diameter of the die, the viscosity of the protein solution, and the like.For example, in the case of a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm, spinning is performed. From the viewpoint of the stability, the extrusion speed is preferably 0.2 to 6.0 mL / h per hole, more preferably 1.4 to 4.0 mL / h per hole. The length of the desolvation tank (coagulation liquid tank) for storing the coagulation liquid is not particularly limited, but may be, for example, 200 to 500 mm. The drawing speed of the undrawn yarn formed by coagulation of the protein may be, for example, 1 to 14 m / min, and the residence time may be, for example, 0.01 to 0.15 min. The take-up speed of the undrawn yarn is preferably 1 to 3 m / min from the viewpoint of desolvation efficiency. The undrawn yarn formed by coagulation of protein may be further drawn (pre-drawn) in solid-liquid, but from the viewpoint of suppressing evaporation of lower alcohol used in the coagulation liquid, the coagulation liquid is kept at a low temperature, It is preferable to take out the coagulated liquid in the state of a drawn yarn.

(B) Stretching The method may further include a step of further stretching the undrawn yarn obtained by the method described above. The stretching may be a single-stage stretching or a multi-stage stretching of two or more stages. When stretched in multiple stages, molecules can be oriented in multiple stages and the total stretch ratio can be increased, so that it is suitable for production of a fiber having high toughness.

  When the protein molded product is a film (protein film), the protein solution may be adjusted to a concentration and a viscosity that can form a film, if necessary. The method of forming the protein into a film is not particularly limited, but the protein solution is applied to an acid-resistant flat plate to a predetermined thickness to form a coating film, and the solvent is removed from the coating film to remove the solvent. And a method of obtaining a film having a thickness of

  As a method of forming a film having a predetermined thickness, for example, a casting method can be mentioned. When a film is formed by a casting method, a protein solution is cast on a flat plate to a thickness of several microns or more using a jig such as a doctor coater or a knife coater to form a cast film, and then dried under reduced pressure or desolventized. A protein film (polypeptide film) can be obtained by removing the solvent by immersion in a tank. The protein film can be formed according to the method described in Japanese Patent No. 5678283.

  When the protein molded body is a porous body (protein porous body), the concentration and the viscosity may be adjusted as necessary so that the protein can be made porous. The method for forming the protein porous body is not particularly limited. For example, a method for obtaining a porous body by adding an appropriate amount of a foaming agent to a protein solution adjusted to a concentration and a viscosity suitable for making porous and removing the solvent, or a method described in Japanese Patent No. 5796147. And the like.

  When the protein molded article is a molded article (protein molded article), the method for forming the protein molded article is not particularly limited. For example, by introducing a dry protein powder into a pressure molding machine and then performing pressure and heating using a hand press or the like, the temperature of the dried protein powder reaches a required temperature, and a molded article can be obtained. The formation of the protein molded article can be performed according to a method described in a patent (Japanese Patent Application No. 2017-539869, PCT / JP2016 / 076500).

  When the protein molded article is a gel molded article (protein gel molded article), the method for forming the protein gel molded article is not particularly limited. For example, a gel formed body can be obtained by a solution generation step of dissolving a dried protein in a solvent for dissolution to obtain a polypeptide solution and a step of replacing the solution generated in the solution generation step with a water-soluble solvent. At this time, between the solution generating step and the step of replacing the dissolving solvent with a water-soluble solvent, a step of pouring into a mold and molding into a predetermined shape is inserted, or the solvent for dissolving is replaced with a water-soluble solvent. A predetermined shape can be obtained by cutting after the process. The formation of the protein gel molded article can be performed according to the method described in Japanese Patent No. 05782580.

When the protein compact is a powder (protein powder compact), the method for forming the powder is not particularly limited.

(Acid or base)
The acid releaser according to the present embodiment contains an acid or a base in addition to the protein composition described above. The acid or base functions as a catalyst for the hydrolysis reaction of the esterified protein, and releases an acid (carboxylic acid) by hydrolysis of the ester group.

  The acid is not particularly limited, and may be either an inorganic acid or an organic acid. Examples of the inorganic acid include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid and the like. Examples of the organic acid include a carboxylic acid and a sulfonic acid. As the acid, a carboxylic acid is preferred. The carboxylic acid used in the process of producing the esterified protein may remain in trace amounts on the surface or inside of the protein composition. By selecting a carboxylic acid as the acid, the remaining carboxylic acid can be used as it is.

  As the carboxylic acid, for example, formic acid, acetic acid, dichloroacetic acid, trifluoroacetic acid, propionic acid, butanoic acid, isobutyric acid, pentanoic acid, caproic acid, caprylic acid, capric acid, monocarboxylic acids such as benzoic acid, capric acid, Saturated aliphatic carboxylic acids such as lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, montanic acid, melissic acid and celloplastic acid, undecylenic acid, oleic acid, elaidic acid, setrein Acids, erucic acid, brassic acid, sorbic acid, linoleic acid, linolenic acid, arachidonic acid, unsaturated aliphatic carboxylic acids such as propiolic acid and stearic acid, and oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid , Pimelic acid, suberic acid, azelaic acid, sebacine , Undekanin acid, Burashirin acid, maleic acid, and dicarboxylic acids such as fumaric acid, and glutaconic acid. The carboxylic acid may be in the form of an acid anhydride or an acid chloride.

  Examples of the sulfonic acid include methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

  The base is not particularly limited as long as it is soluble in water, and may be either an inorganic base or an organic base. Examples of the inorganic base include potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, sodium carbonate and the like. Examples of the organic base include primary amines such as alkylamines such as ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine and triethylamine, aminoethanol, methylaminoethanol, dimethylaminoethanol, ethylaminoethanol, diethylaminoethanol and diethanolamine. And tertiary amines.

(Method for producing acid releaser)
The acid releaser according to the present embodiment can be obtained by including the above-mentioned acid or base in the above-mentioned protein composition. The acid or base may be added to the protein composition, or the acid or base remaining on the surface or inside of the protein composition (part of the acid or base used in the process of producing the protein composition) You may use it as it is. In addition, since an acid or a base functions as a catalyst of the ester hydrolysis reaction, it may be present in an extremely small amount.

(Use of acid emitter)
The acid releasing body according to the present embodiment can release a carboxylic acid by contacting with water and hydrolyzing an ester group. At this time, the acid or base contained in the acid releaser acts as a catalyst for hydrolysis of the ester group.

  In the acid releasing body according to the present embodiment, the rate of acid release can be controlled by changing at least one of the temperature of the acid releasing body and the temperature of moisture to be brought into contact. That is, at lower temperatures the rate of acid release decreases, and at higher temperatures the rate of acid release increases. As the temperature is lower, a longer acid releasing effect can be expected, and as the temperature is higher, a higher acid releasing effect can be expected in a certain time.

  The acid-releasing body according to the present embodiment exhibits a bactericidal effect, a deodorant effect, a deodorant effect, and the like by the released acid, and thus can be used as a bactericide, a preservative, a deodorant, and the like. In this case, the esterified protein preferably contains at least one selected from the group consisting of formate, acetate and propionate from the viewpoint of the bactericidal effect. In addition, the acid-releasing body according to the present embodiment exhibits an anti-mite effect, a vermin-repelling effect, and the like by increasing the acid release rate, and thus can be used as an insect repellent.

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

[(1) Preparation of target protein-expressing strain (recombinant cell)]
A nucleic acid encoding a modified spider silk fibroin (PRT799) having the amino acid sequence represented by SEQ ID NO: 15 was synthesized. The nucleic acid was added with an NdeI site at the 5 ′ end and an EcoRI site downstream of the stop codon.

  Each of the above nucleic acids was cloned into a cloning vector (pUC118). Then, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector. Escherichia coli BLR (DE3) was transformed with the pET-22b (+) expression vector obtained by recombining the nucleic acid to obtain a transformed Escherichia coli (recombinant cell) expressing the target protein.

[(2) Expression of target protein]
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 4) so that the OD ₆₀₀ became 0.005. The temperature of the culture was maintained at 30 ° C., and the flask was cultured until the OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture.

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

  Immediately after glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, 120 g / 1 L of yeast extract) was added at a rate of 1 mL / min. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. The culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the target protein. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the target protein was expressed as an insoluble substance due to the appearance of a band of the target protein size dependent on the addition of IPTG. Confirmed that.

[(3) Purification of protein]
Two hours after the addition of IPTG, the recovered cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in an 8 M guanidine buffer (8 M 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. Stirred for minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after the dialysis was collected by centrifugation, the water was removed with a freeze dryer, and the freeze-dried protein powder (PRT799) was collected.

[(4) Molding of protein fiber]
A dry powder of the modified spider silk fibroin (PRT799) was dissolved in formic acid to obtain a dope solution (protein concentration in the dope solution: 25% by mass). Using a known spinning device, the dope solution was discharged into a coagulating solution (methanol) by a gear pump. The spinning conditions were as shown below. Thus, a protein fiber (fibroin fiber) was obtained as a protein molded body. In the obtained protein fiber, a small amount of formic acid, which is a solvent of the dope solution, remains.
(Spinning conditions)
Dope temperature: 25 ° C
Hot roller temperature: 60 ° C

[(5) Hydrolysis by steam]
The protein fibers were allowed to stand still under the conditions of a humidity of 80% and 60 ° C., and the absorbance ratio P1 / P2 was measured every day using FT-IR (FT-IR iS50, manufactured by NICOLET).

[(6) Confirmation of formate group reduction behavior by FT-IR]
The fiber subjected to the hydrolysis treatment and dried was measured by the FT-IR microscopic transmission method to confirm the behavior of reducing the formate group. For each sample treated under each pH condition, the peak height ratio P1 / P2 was determined and recorded. Table 6 shows the results. P1 and P2 are peak heights corresponding to the following wave numbers, and a smaller value of the absorbance ratio P1 / P2 means that the number of structural ester groups is smaller.
P1: 1725cm- ¹ peak height (peak based on C = O of ester) P2: 1445cm- ¹ peak height (peak based on the amide III protein)

  FIG. 6 is a diagram plotting the data of Table 6 with the absorbance ratio P1 / P2 on the vertical axis and the processing time (unit: days) on the horizontal axis.

  As shown in Table 6 and FIG. 6, when the protein fibers were brought into contact with moisture, the absorbance ratio decreased. The results indicate that the ester hydrolysis reaction proceeds with the contact of moisture with the remaining formic acid as a catalyst, and the ester groups added to the protein fibers decrease. Further, even after 7 days from the contact with water, the absorbance ratio was a value equal to or higher than the detection limit. This result indicated that the ester group added to the protein fiber remained for a long period of time and the acid releasing ability was maintained.

Claims (6)

  An acid emitter comprising a protein composition comprising an esterified protein and an acid or base.   The acid emitter according to claim 1, wherein the protein is a structural protein.   3. The acid emitter of claim 2, wherein said structural protein is a modified fibroin.   The acid emitter according to claim 3, wherein the modified fibroin is a modified spider silk fibroin.   The acid releaser according to any one of claims 1 to 4, wherein the protein composition is at least one selected from the group consisting of a fiber, a film, a gel, a porous body, and a molded article.   The acid releaser according to any one of claims 1 to 5, wherein the esterified protein contains at least one selected from the group consisting of formate, acetate, and propionate.

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