JP2020026409A - Protein molding subjected to high-pressure treatment after molding - Google Patents

Abstract

To provide a method for improving the physical properties of a protein molding without considering complicated conditions, an article having improved physical properties and a method of producing the same.SOLUTION: A method for producing a compressed body of a protein molding has a step of pressurizing a protein molding obtained by molding protein.SELECTED DRAWING: None

Description

  The present invention relates to a protein molded product subjected to high pressure treatment after molding.

  Conventionally, as a method for improving the physical properties of a protein molded product obtained by molding a protein, for example, when the shape of the protein molded product is a yarn, a method of optimizing spinning conditions is known (for example, Patent Document 1).

International Publication No. 2012/165577

  However, in the technique described in Patent Document 1, it is necessary to set detailed conditions in accordance with the shape of the protein molded product, the amino acid sequence used as the raw material, and the like.

  Therefore, an object of the present invention is to provide a method for improving the physical properties of a protein molded article, an article with improved physical properties, and a method for producing the same, without considering complicated conditions.

The present invention provides the following [1] to [36].
[1] A method for producing a pressurized protein molded body, comprising a step of applying pressure to a protein molded body obtained by molding a protein.
[2] The method for producing a pressurized body according to [1], wherein the step of applying pressure to the protein molded body does not stretch the protein molded body.
[3] The method for producing a pressurized body according to [1] or [2], wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body from at least two directions.
[4] The method for producing a pressurized body according to any one of [1] to [3], wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body via a liquid.
[5] The method for producing a pressurized body according to any one of [1] to [4], wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body in a liquid.
[6] The method for producing a pressurized body according to [4] or [5], wherein the liquid contains a polar solvent.
[7] The method for producing a pressurized body according to [6], wherein the polar solvent contains an alcohol.
[8] The method for producing a pressurized body according to [7], wherein the alcohol is ethanol.
[9] The method for producing a pressurized body according to any one of [4] to [8], wherein the liquid contains an additive.
[10] The method for producing a pressurized body according to [9], wherein the additive is a crosslinking agent or an antioxidant.
[11] The method for producing a pressurized body according to any one of [1] to [10], wherein the applied pressure is 50 MPa or more.
[12] The method for producing a pressurized body according to any one of [1] to [11], wherein the protein molded body is immersed in the liquid for at least 24 hours before applying pressure.
[13] The method for producing a pressurized body according to [12], wherein the liquid is alcohol.
[14] The method for producing a pressurized body according to [13], wherein the alcohol is ethanol.
[15] The method for producing a pressurized body according to any one of [1] to [14], wherein the protein molded body is in the form of a thread or a film.
[16] The method for producing a pressurized body according to any one of [1] to [15], wherein the protein is a structural protein.
[17] The method for producing a pressurized body according to [16], wherein the structural protein is a spider silk protein.
[18] A processing method for improving the physical properties of a protein molded product, comprising a step of applying pressure to a protein molded product obtained by molding a protein.
[19] The method according to [18], wherein the step of applying pressure to the protein molded body does not stretch the protein molded body.
[20] The method according to [18] or [19], wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body from at least two directions.
[21] The method according to any one of [18] to [20], wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body via a liquid.
[22] The method according to any one of [18] to [21], wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body in a liquid.
[23] The method for producing a pressurized body according to [18] or [21], wherein the liquid contains a polar solvent.
[24] The method according to [23], wherein the polar solvent comprises an alcohol.
[25] The method according to [24], wherein the alcohol is ethanol.
[26] The method according to any one of [21] to [25], wherein the liquid contains an additive.
[27] The method according to [26], wherein the additive is a crosslinking agent or an antioxidant.
[28] The method according to any one of [18] to [27], wherein the applied pressure is 50 MPa or more.
[29] The method according to any one of [18] to [28], wherein the protein molded body is immersed in the liquid for at least 24 hours before applying pressure.
[30] The method according to any one of [18] to [29], wherein the protein molded product is in the form of a thread or a film.
[31] The method according to any one of [18] to [30], wherein the protein is a structural protein.
[32] The method according to any one of [18] to [31], wherein the structural protein is a spider silk protein.
[33] A method for producing a immersed protein molded article, comprising a step of immersing a protein molded article obtained by molding a protein in alcohol.
[34] The method for producing a immersed protein molded product according to [33], wherein the alcohol is ethanol.
[35] A processing method for improving physical properties of a protein molded article, comprising a step of immersing a protein molded article obtained by molding a protein in alcohol.
[36] The method for treating a protein molded article according to [35], wherein the alcohol is ethanol.

  Advantageous Effects of Invention According to the present invention, it is possible to provide a pressed body of a protein molded body having improved physical properties by a simple operation without considering complicated conditions. The pressurized body provided by the present invention has biodegradability and is superior in stress, elongation, and toughness as compared to a protein molded body before pressurization.

It is the schematic which shows an example of the spinning apparatus for manufacturing a modified spider silk fibroin fiber. It is a photograph which shows the state which enclosed the protein molded object and the liquid in the flexible container. It is a schematic diagram which shows an example of the process of applying pressure to a protein molded object.

  Hereinafter, preferred embodiments of the present invention will be described.

  One embodiment of the present invention is a pressurized body of a protein molded product obtained by molding a protein. The protein molded body can be obtained by molding a protein, and the pressurized body can be produced by a method including a step of applying pressure to the protein molded body. The method according to the present embodiment also includes a method for producing a pressurized body of a protein molded body, which includes a step of molding a protein to obtain a protein molded body, and a step of applying pressure to the protein molded body.

[protein]
The protein is not particularly limited, and may be produced by a microorganism or the like by genetic recombination technology, may be chemically synthesized, or may be obtained by purifying a naturally occurring protein. It may be something.

  As used herein, the term "comprising as a main component" means that at least 50% by mass of the total mass of the protein fiber is protein. The mass ratio of the protein in the protein fiber may be 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, and 90% by mass or more.

  The protein may be, for example, a structural protein or an artificial protein derived from the structural protein. A structural protein refers to a protein that forms or retains a structure, form, and the like in a living body. Examples of the structural protein include fibroin, keratin, collagen, elastin, resilin and the like. Preferred proteins are fibroin or artificial proteins derived from fibroin. One of the above-mentioned structural proteins and artificial proteins derived from the structural proteins can be used alone or in combination of two or more.

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

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

  The spider silk fibroin may be a natural spider silk protein or an artificial protein derived from a natural spider silk protein.

  Natural spider silk proteins include, for example, large spinal canal thread proteins, weft proteins, and small bottle gland proteins. Since the large spinal cord marker thread protein has a repetitive region consisting of a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity. On the other hand, the weft protein has a feature that it does not have a crystalline region but has a repeating region composed of an amorphous region. The weft protein has a lower stress but a higher elasticity than the large spinal canal thread protein.

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

  Weft protein is produced in the flagellar gland of spiders. Examples of the weft protein include a flagellaform silk protein derived from the American spider spider (Nephila clavipes).

  An artificial protein derived from a natural spider silk protein may be a recombinant spider silk protein. Examples of the recombinant spider silk protein include a mutant, analog or derivative of a natural spider silk protein. One suitable example of such an artificial protein is a recombinant spider silk protein of a large spinal canal silk protein. For example, recombinant spider silk proteins have been produced in several heterologous protein production systems, and their production methods use transgenic goats, transgenic silkworms, or recombinant plant or mammalian cells.

The recombinant spider silk protein can be obtained, for example, by deleting one or more of the sequences encoding the (A) _n motif from the cloned natural fibroin gene sequence. Further, for example, it is obtained by designing an amino acid sequence corresponding to deletion of one or more (A) _n motifs from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. You can also. In any case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. Amino acid sequence modification corresponding to the above may be performed. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), and Methods in Enzymology, 100, 448 (1983).

Examples of the artificial spider silk protein derived from the silkworm silk protein and the silkworm silk protein derived from silkworm silk include a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m. Can be Here, in Formula 1, in the (A) _n motif, A represents an alanine residue, and n represents 2 to 27, 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to It may be an integer of 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, 86% or more. % Or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. m shows the integer of 2-300. The total number of glycine (Gly), serine (Ser) and alanine (Ala) residues contained in the amino acid sequence represented by Formula 1 is preferably at least 40%, more preferably at least 60%, based on the total number of amino acid residues. Or it may be 70% or more. 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 artificial protein derived from the large spinal cord marker thread include a protein containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

Examples of the protein derived from the weft protein include, for example, a protein containing a domain sequence represented by Formula 2: [REP2] _o (wherein, REP2 is composed of Gly-Pro-Gly-Gly-X X represents an amino acid sequence, and X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr), and valine (Val), and o represents an integer of 8 to 300. Can be mentioned. Examples of the polypeptide derived from the weft protein include a polypeptide containing 10 or more, preferably 20 or more, more preferably 30 or more units of the amino acid sequence represented by the formula 2: REP2. When a recombinant protein is produced from a weft protein using a microorganism such as Escherichia coli as a host, the molecular weight is preferably 500 kDa or less, more preferably 300 kDa or less, and still more preferably, from the viewpoint of productivity. Is 200 kDa or less. Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 3. The amino acid sequence represented by SEQ ID NO: 3 is 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 number: AAF36090, GI: 7106224). The amino acid sequence from residues 1220 to 1659 (referred to as PR1 sequence) and the partial sequence of the flagellar silk protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649) A C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminus is joined, and the amino acid sequence represented by SEQ ID NO: 4 (tag sequence and hinge sequence) is added to the N-terminus of the joined sequence; is there.

As a protein derived from collagen, for example, a protein containing a domain sequence represented by Formula 3: [REP3] _p (where p represents an integer of 5 to 300. In Formula 3, REP3 is Gly-X- An amino acid sequence composed of Y is shown, and X and Y each represent an arbitrary amino acid residue other than glycine (Gly). A plurality of REP3s may have the same amino acid sequence or different amino acid sequences.) Can be mentioned. Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 5. The amino acid sequence represented by SEQ ID NO: 5 corresponds to a repeat portion and a motif of a partial sequence of human collagen type 4 obtained from the NCBI database (Accession number of GenBank of NCBI: CAA56335.1, GI: 3702452). The amino acid sequence represented by SEQ ID NO: 6 (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 4: [REP4] _q (where, in Formula 4, q represents an integer of 4 to 300. REP4 is Ser-JJ) -Represents an amino acid sequence composed of -Tyr-Gly-U-Pro, wherein J represents an arbitrary amino acid residue, particularly an amino acid selected from the group consisting of aspartic acid (Asp), serine (Ser) and threonine (Thr); U is an arbitrary amino acid residue, particularly an amino acid residue selected from the group consisting of proline (Pro), alanine (Ala), threonine (Thr) and serine (Ser). A plurality of REP4s may have the same amino acid sequence or different amino acid sequences.) You. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 7 can be mentioned. The amino acid sequence represented by SEQ ID NO: 7 replaces the threonine (Thr) at the 87th residue with serine (Ser) in the amino acid sequence of resilin (NCBI GenBank Accession No. NP 611157, Gl: 246654243), and The amino acid sequence represented by SEQ ID NO: 12 (tag sequence) is located at the N-terminus of the amino acid sequence from residue 19 to residue 321 of the sequence obtained by substituting asparagine (Asn) at the 95th residue with aspartic acid (Asp). It has been added.

  Examples of proteins derived from elastin include proteins having an amino acid sequence such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine), and the like. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 8 can be mentioned. The amino acid sequence represented by SEQ ID NO: 8 corresponds to the amino acid sequence represented by SEQ ID NO: 6 (tag sequence) at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of NCBI GenBank Accession No. AAC98395. And a hinge sequence).

  Examples of keratin-derived proteins include Capra hircus type I keratin. Specific examples include a protein comprising the amino acid sequence represented by SEQ ID NO: 9 (the amino acid sequence of GenBank Accession No. ACY30466 of NCBI).

(Method for producing artificial protein)
The protein contained in the protein fiber is, for example, a host transformed with an expression vector having a nucleic acid sequence encoding a desired protein and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing a nucleic acid.

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

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

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

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

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

  When a prokaryote is used as a host, as a vector for introducing a nucleic acid encoding a target protein, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.

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

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

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

  The 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.

  The desired recombinant 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 salts, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

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

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

  The recombinant protein produced by the transformed host can be isolated and purified by a method generally used for isolating and purifying proteins. 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.

  As the above-mentioned chromatography, column chromatography using phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), and Sephadex G-150 (Pharmacia Biotech) is preferably used.

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

<Method for producing protein molded article>
The shape of the protein molded body is not particularly limited, and powder, long fiber, short fiber, monofilament, multifilament, spun yarn, twisted yarn, processed yarn, mixed fiber, mixed spun yarn, nonwoven fabric, cotton (cotton), plate, It may be in the form of a film, sheet or the like.

(1) Method for Producing Protein Film A film-shaped protein molded product (protein film) can be produced, for example, by the following procedure. That is, the obtained protein was dissolved in a solvent, a stabilizer was added, if necessary, and the mixture was stirred to prepare a dope solution. The prepared dope solution was cast on a petri dish made of, for example, polystyrene (PS), and dried by allowing it to stand. Then, the dried product of the cast dope solution is immersed in a mixed solution of acetone and methanol (for example, a volume ratio of 7: 3) together with the Petri dish, and the film is peeled off from the Petri dish. A protein film is prepared by immersing the peeled film in a methanol solution and performing natural drying.

(2) Method for Producing Protein Fiber A fibrous protein molded article (protein fiber) can be produced by a known spinning method. That is, for example, when producing a protein fiber containing a protein as a main component, first, the protein produced according to the above-described method is converted into dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, or If necessary, a dope solution is prepared by adding to a solvent such as hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution promoter and dissolving the same. Next, protein fibers can be obtained by spinning the dope using a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning. Preferred spinning methods include wet spinning and dry-wet spinning.

  FIG. 1 is a schematic diagram showing an example of a spinning device for producing protein fibers. A spinning device 10 shown in FIG. 1 is an example of a spinning device for dry-wet spinning, and includes an extruder 1, an undrawn yarn manufacturing device 2, a wet heat drawing device 3, and a drying device 4.

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

  The coagulation liquid 11 may be any solvent that can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulating liquid 11 may appropriately contain water. The temperature of the coagulating liquid 11 is preferably 0 to 30C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the base 9, the extrusion speed is preferably 0.2 to 6.0 mL / hour per hole, and 1.4 to 4.0 mL / hour. More preferably, it is time. The distance over which the coagulated modified fibroin passes through the coagulating liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) may be long enough to efficiently remove the solvent. 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, preferably 1 to 3 m / min. The residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and is preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11. The coagulation liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as needed.

  The stretching performed when obtaining the protein fiber may be, for example, dry stretching in addition to wet stretching performed in the above-described coagulation bath 20 and stretching bath 21.

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

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

  The wet heat stretching and the dry heat stretching may each be performed alone, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and further the third stage stretching is performed by dry heat stretching. For example, wet heat stretching and dry heat stretching can be performed in an appropriate combination.

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

<Modified fibroin fiber>
The protein fiber according to the present embodiment may be a fiber obtained by spinning the above-described modified fibroin (also referred to as “modified fibroin fiber”). In this case, the above-described modified fibroin is contained as a main component.

The shrinkage of the modified fibroin fiber is defined by the following equation.
Shrinkage = {1− (length of protein fiber after shrinkage step including contact with water below boiling point / length of protein fiber before shrinkage step)} × 100 (%)

  The modified fibroin fiber according to the present embodiment preferably has a shrinkage ratio of more than 7% when brought into contact with water, more preferably 15% or more, even more preferably 25% or more, It is still more preferably at least 32%, even more preferably at least 40%, particularly preferably at least 48%, particularly preferably at least 56%, and at least 64%. Is even more preferably, and most preferably 72% or more. The upper limit of the shrinkage is usually 80% or less.

<Protein yarn>
The thread-like protein molded product (protein yarn) is, for example, a single yarn that is any one of a twisted yarn containing modified fibroin fibers, a false twisted yarn containing modified fibroin fibers, and a spun yarn containing modified fibroin fibers. A protein yarn, which is a raw yarn used for knitting a knitted fabric or weaving a woven fabric, mainly consists of modified fibroin fibers. The protein thread may consist of modified fibroin fibers. In this case, the protein thread contains the modified fibroin as a main component. The raw yarn used for knitting the knitted fabric may partially include other fibers.

  The raw yarn may be a twisted or false twisted filament (long fiber). The raw yarn may be a spun yarn made of staples (short fibers). The raw yarn used for knitting the knitted fabric in the present embodiment is a single yarn. By using a single yarn, a texture that cannot be obtained with a twin yarn can be realized in a knitted fabric.

  The twist direction of the twisted yarn, the false twisted yarn, or the spun yarn may be S twist (right twist) or Z twist (left twist).

  In the present specification, the occurrence of skew in a knitted fabric or a woven fabric or that is sufficiently suppressed is referred to as "no skew", such a knitted fabric is referred to as "no skew knitted fabric", Such a fabric is referred to as "non-skew fabric". In addition, the distortion of the woven fabric refers to a defective state similar to skew in the woven fabric. The presence or absence of skew or distortion can be determined by visual inspection of the stitch or weave.

  The fineness of the twisted or false-twisted yarn or spun yarn is preferably a gauge of a knitting machine to be used or a suitable count of a loom, and spinning is performed in accordance therewith.

  A single yarn having a count of 13 to 150 Nm is finer than a twin yarn or the like, and has an excellent texture and softness. In addition, the yarn count of the single yarn is not particularly limited, and may be any count obtained by a known spinning method. The number of twists is not particularly limited, and may be any number of twists used in known spinning.

<Immersion treatment>
The protein molded body may be subjected to a pressure treatment after being immersed in the liquid. By immersing the protein molded body in the liquid, the physical properties (eg, stress, elongation, toughness) of the molded body can be improved.

  The liquid is not particularly limited as long as it is a polar solvent, and examples thereof include a protic polar solvent and an aprotic polar solvent. Examples of the protic polar solvent include water, methanol, ethanol, 1-propanol, 2-propanol (isopropanol), butanol, tert-butanol, ethylene glycol, propylene glycol, and glycerin. Examples of the aprotic polar solvent include acetone, ethyl methyl ketone, N, N-dimethylformamide, N, N-acetamide, and dimethyl sulfoxide. The preferred polar solvent is ethanol. By immersion treatment of the protein molded body, elongation and toughness can be particularly improved.

  The liquid may contain additives such as a crosslinking agent and an antioxidant. By immersing in a liquid containing these additives, the physical properties (eg, stress, elongation, toughness) of the molded article can be improved even when the pressure applied in the pressure treatment is lower.

  Examples of the crosslinking agent include DTMMM (4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride) and EDC (1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide hydrochloride), DCC (N, N'-dicyclohexylcarbodiimide, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide), DIC (diisopropylcarbodiimide) and the like.

  Examples of the antioxidant include BHT (2,6-di-tert-butyl-p-cresol), propyl gallate, BHA (butylated hydroxyanisole), TBHQ (tert-butylhydroquinone) and the like.

<Pressure treatment>
The pressurized body of the protein molded body according to the present embodiment can be manufactured by applying pressure to the protein molded body. The pressurizing means of the pressurizing treatment preferably presses the protein molded body from at least two directions. At least two directions for applying the pressure may be two directions facing each other. Examples of the pressurizing means include applying pressure to the protein molded body by applying pressure to the liquid while the protein molded body is immersed in the liquid. In addition, in the pressing treatment, it is not necessary to stretch the protein molded body while applying pressure.

  As a preferable pressurizing means, for example, as shown in FIG. 2, the protein molded body is sealed together with a liquid in a flexible container such as a film bag, and pressure is applied from the outside of the flexible container. is there. More specifically, for example, as shown in FIG. 3, a flexible container in which a pressure container 61 of a pressurizing device 60 provided with a pressure sensor is filled with a pressurizing liquid 62, and a protein molded body 65 and a liquid 64 are sealed. A method may be used in which 63 is immersed in the pressurizing liquid 62 to apply pressure.

  The lower limit of the applied pressure is not particularly limited, and may be, for example, 25 MPa or more, 50 MPa or more, 100 MPa or more, 200 MPa or more, or 600 MPa or more. The upper limit of the applied pressure is not particularly limited, and may be, for example, 800 MPa or less, 200 MPa or less, or 100 MPa or less. Particularly preferred pressures are 50-800 MPa, 50-600 MPa, or 100-600 MPa.

  The flexible container may be any as long as it does not break at the pressure of the pressure treatment and can sufficiently transmit the external pressure into the container. Examples of the material of the flexible container include polypropylene.

  When the liquid in the flexible container contains an additive, the lower limit of the applied pressure is not particularly limited, and may be, for example, 25 MPa or more, 50 MPa or more, 100 MPa or more, 200 MPa or more, or 600 MPa or more. Good. The upper limit of the pressure applied in this case may be 800 MPa or less, 200 MPa or less, or 100 MPa or less. Particularly preferred pressures are 50-800 MPa, 50-600 MPa, or 100-600 MPa.

  The time for applying pressure can be appropriately set, and may be, for example, 1 hour, 0.5 minutes, 3 minutes, 5 minutes, or 10 minutes.

  Further, the pressure treatment may be performed in two or more stages. The pressure applied in the pressure treatment may be higher or lower than the pressure applied in the previous pressure treatment. For example, the pressure treatment may be performed in multiple stages so that the applied pressure gradually increases. Specifically, in the first pressurizing process, a pressure of 50 MPa may be applied for a predetermined time, and in the second pressurizing process, a pressure of 600 MPa may be applied for a predetermined time.

  Another embodiment of the present invention is a treatment method for improving the physical properties of a protein molded product, including a step of applying pressure to a protein molded product obtained by molding a protein. The protein, the protein molded body, and the step of applying pressure in the present embodiment are as described above.

  The physical properties in the present specification are physical properties of a protein molded article, and more specifically, mean stress, elongation, and toughness. In the present specification, “improving physical properties” means that at least one of stress, elongation, and toughness is improved as compared with a value of a protein molded body before pressure is applied.

  Another embodiment of the present invention is a pressurized body of a protein molded body obtained by molding a protein, the pressurized body having an improved birefringence.

  The birefringence is one of data used as an index of the orientation of a protein. By applying pressure to the protein molded body, the orientation of the protein is further improved as compared with the protein molded body before the pressure is applied.

  The birefringence of the pressurized body of the protein molded body according to the present embodiment can be improved by applying pressure, as compared with the protein molded body before pressurization. As the birefringence increases, the stress, elongation and toughness tend to increase.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples.

<Production of modified spider silk fibroin>
(1) Preparation of plasmid expression strain Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank Accession No .: P468804.1, GI: 11744415) derived from Nephila clavipes, the amino acid sequence represented by SEQ ID NO: 11 The modified fibroin (hereinafter, also referred to as “PRT799”) was designed. The amino acid sequence represented by SEQ ID NO: 11 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues with respect to the amino acid sequence of fibroin derived from Nephila clavipes for the purpose of improving productivity. Further, an amino acid sequence represented by SEQ ID NO: 6 (tag sequence and hinge sequence) is added to the N-terminus.

  Next, a nucleic acid encoding PRT799 was synthesized. An NdeI site at the 5 'end and an EcoRI site downstream of the stop codon were added to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). 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.

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence represented by SEQ ID NO: 10. 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 1) so that the OD ₆₀₀ was 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 a production medium (Table 2) 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. Further, the concentration of dissolved oxygen in the culture solution 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. Further, the culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 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 carried out using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the desired spider silk fibroin due to the appearance of the target spider silk fibroin size band depending on the addition of IPTG. It was confirmed.

(3) Purification of protein The cells collected 2 hours after the addition of IPTG were washed with a 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. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after the dialysis was collected by centrifugation, water was removed with a freeze dryer, and the freeze-dried powder was collected to obtain a modified spider silk fibroin “PRT799”.

<Preparation of modified spider silk fibroin fiber>
(1) Preparation of Dope Solution After the above-mentioned modified spider silk fibroin (PRT799) was added to dimethyl sulfoxide (DMSO) at a concentration of 24% by mass, LiCl was dissolved at a concentration of 4.0% by mass as a dissolution promoter. Was added. Then, using a shaker, the modified spider silk fibroin was dissolved over 3 hours to obtain a DMSO solution. Dust and bubbles in the obtained DMSO solution were removed to prepare a dope solution. The viscosity of the dope solution was 5000 cP (centipoise) at 90 ° C.

(2) Spinning Using the spinning apparatus 10 shown in FIG. 1, the obtained dope liquid was discharged into a coagulation liquid (methanol), stretched, and dried to obtain a filament of PRT799. The obtained filament of PRT799 was wound around a bobbin.
Temperature of coagulation liquid (methanol): 5 to 10 ° C
Stretching ratio: 4.52 times Drying temperature: 80 ° C

<Immersion treatment of molded body>
(1) Immersion treatment conditions The filament of PRT799 was cut into 100 cm and immersion treatment was performed. The immersion treatment was performed by enclosing the immersion solvent and the filament in a polypropylene bag as shown in FIG.

(2) Filament tensile test The physical properties (stress, elongation, toughness) of the filament of PRT799 were measured. Specifically, the filament was gripped and fixed to a test paper strip with an adhesive at a jig distance of 20 mm, and a tensile tester (manufactured by Instron, model: 3342) was used at a temperature of 20 ° C. and a relative humidity of 65%. The stress (strength) and elongation were measured at a tensile speed of 10 cm / min. In the test, the load cell capacity was set to 10 N, and a clip jig was used.

(3) Measurement results Table 3 shows the evaluation results. The values of stress, elongation, and toughness of Reference Example 2 were shown as relative values when the value of Reference Example 1 was set to 100. By immersing the filament of PRT799 in ethanol for 48 hours, the elongation and toughness were improved as compared with the filament not immersed.

<Pressure treatment of molded body-1>
(1) Pressure treatment condition The filament of PRT799 is cut into 100 cm, and pressure treatment is performed at a predetermined pressure for 10 minutes using an ultrahigh pressure hydrostatic pressure treatment device (manufactured by Sugino Machine Co., Ltd., model: HPV-80C20-SA). Was done.
Water or ethanol was used as the immersion solvent, and in some examples, DTMMM (crosslinking agent, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholine Ium chloride) and BHT (antioxidant, 2,6-di-tert-butyl-p-cresol) were added to the solvent. The immersion time of “0 hours” means that the pressure treatment was performed immediately after the immersion solvent and the filament were sealed in the bag.

(2) Filament tensile test The physical properties (stress, elongation, toughness) of the filament of PRT799 were measured. Specifically, the filament was gripped and fixed to a test paper strip with an adhesive at a jig distance of 20 mm, and a tensile tester (manufactured by Instron, model: 3342) was used at a temperature of 20 ° C. and a relative humidity of 65%. The stress (strength) and elongation were measured at a tensile speed of 10 cm / min. In the test, the load cell capacity was set to 10 N, and a clip jig was used.

(3) Measurement results The evaluation results are shown in Tables 4 and 5. The values of stress, elongation, and toughness of Example 1 were shown as relative values when the value of Comparative Example 1 was set to 100. The values of stress, elongation, and toughness of Examples 2 to 11 are shown as relative values when the value of Comparative Example 2 is set to 100. In any of the examples, by performing the pressure treatment, the strength and elongation of the filament were improved. In addition, when pressure treatment was performed by immersion in ethanol containing an additive (Examples 10, 11, and 12), the elongation and toughness were significantly improved even at a lower pressure (50 MPa).

<Pressure treatment of molded body -2>
(1) Pressure treatment condition The filament of PRT799 is cut into 100 cm, and pressure treatment is performed at a predetermined pressure for 10 minutes using an ultrahigh pressure hydrostatic pressure treatment device (manufactured by Sugino Machine Co., Ltd., model: HPV-80C20-SA). Was done.
Ethanol was used as the immersion solvent, and in some examples, BHT (antioxidant, 2,6-di-tert-butyl-p-cresol) was added to the solvent. The immersion time of “0 hours” means that the pressure treatment was performed immediately after the immersion solvent and the filament were sealed in the bag. In Example 15, a two-stage pressurizing process was performed. After pressurizing at 50 MPa for 10 minutes (first pressurizing process), a pressurizing process was performed at 600 MPa for 10 minutes (second pressurizing process). ).
Immersion time: 0 hours (immediately after encapsulation), 48 hours (after 2 days)

(2) Filament tensile test The physical properties (stress, elongation, toughness) of the filament of PRT799 were measured. Specifically, the filament was gripped and fixed to a test paper strip with an adhesive at a jig distance of 20 mm, and a tensile tester (manufactured by Instron, model: 3342) was used at a temperature of 20 ° C. and a relative humidity of 65%. The stress (strength) and elongation were measured at a tensile speed of 10 cm / min. In the test, the load cell capacity was set to 10 N, and a clip jig was used.

(3) Measurement results Table 6 shows the evaluation results. The numerical values of stress, elongation and toughness in each example are shown as relative values when the numerical value of Comparative Example 1 is set to 100. In any of the examples, by performing the pressure treatment, the strength and elongation of the filament were improved. Further, in Examples 14 and 15 in which the pressure treatment was performed in ethanol containing the additive, the elongation and the toughness were greatly improved even at a lower pressure (50 MPa). In Example 15, the stress was further improved.

<Measurement of birefringence>
The filament of PRT799 was cut into 100 cm, and subjected to a pressure treatment at a predetermined pressure for 10 minutes using an ultrahigh pressure hydrostatic pressure treatment device (manufactured by Sugino Machine Co., model: HPV-80C20-SA). With respect to the obtained filaments of Examples 16 and 17, the birefringence was measured using a microscope (manufactured by OLYMPUS, model: BX51).

Table 7 shows the results. The numerical value of the birefringence index of each example is shown as a relative value when the numerical value of Comparative Example 1 is 100. It was confirmed that the filaments of Examples 16 and 17 had improved birefringence and more advanced orientation than the filaments of Comparative Example 3 which had not been subjected to the immersion treatment and the pressure treatment.

Claims (14)

  A method for producing a pressed body of a protein molded body, comprising a step of applying pressure to a protein molded body obtained by molding a protein.   The method for producing a pressurized body according to claim 1, wherein the step of applying pressure to the protein molded body does not stretch the protein molded body.   The method for producing a pressurized body according to claim 1, wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body from at least two directions.   The method for producing a pressurized body according to any one of claims 1 to 3, wherein the step of applying pressure to the protein molded body applies pressure to the protein molded body via a liquid.   The method for producing a pressurized body according to claim 4, wherein the liquid contains a polar solvent.   The method for producing a pressurized body according to claim 4, wherein the liquid contains an additive.   The method for producing a pressurized body according to any one of claims 1 to 6, wherein the pressure to be applied is 50 MPa or more.   The method for producing a pressurized body according to any one of claims 1 to 7, wherein the protein molded body is immersed in a liquid for 24 hours or more before applying pressure.   The method for producing a pressurized body according to any one of claims 1 to 8, wherein the protein molded body is in the form of a thread or a film.   The method for producing a pressurized body according to any one of claims 1 to 9, wherein the protein is a structural protein.   The method for producing a pressurized body according to claim 10, wherein the structural protein is a spider silk protein.   A processing method for improving physical properties of a protein molded body, comprising a step of applying pressure to a protein molded body obtained by molding a protein.   13. The method of claim 12, wherein applying pressure to the protein compact comprises applying pressure to the protein compact via a liquid. 14. The method according to claim 12, wherein the protein is a spider silk protein.