WO2020017652A1 - Procédé de production de fibre de protéine - Google Patents

Procédé de production de fibre de protéine Download PDF

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Publication number
WO2020017652A1
WO2020017652A1 PCT/JP2019/028547 JP2019028547W WO2020017652A1 WO 2020017652 A1 WO2020017652 A1 WO 2020017652A1 JP 2019028547 W JP2019028547 W JP 2019028547W WO 2020017652 A1 WO2020017652 A1 WO 2020017652A1
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Prior art keywords
protein
solvent
mass
spinning
amino acid
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PCT/JP2019/028547
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English (en)
Japanese (ja)
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弘放 ▲遅▼
▲郁▼▲群▼ ▲荘▼
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Ｓｐｉｂｅｒ株式会社
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    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a method for producing a protein fiber.
  • a wet spinning method and a dry-wet spinning method in which a spinning solution discharged from a nozzle is coagulated in a coagulation bath to form fibers are known.
  • the wet spinning method and the dry-wet spinning method are also used when producing protein fibers containing protein as a main component (for example, see Patent Document 1).
  • a protein solution in which a protein is dissolved in a solvent is used as a dope solution (spinning solution), and the dope solution is coagulated from a spinneret into a desolvation bath.
  • a protein fiber is obtained by extruding a solvent from a dope solution and forming a fiber to form an undrawn yarn (for example, see Patent Document 2).
  • undrawn yarn of synthetic fibers is thick, and its cross-sectional shape is easily broken.
  • Known solvents for dissolving proteins include dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, and formic acid.
  • lower alcohols such as methanol, ethanol, and 2-propanol are widely used as coagulation bath solutions in order to remove the solvent and form fibers.
  • unstretched yarns are not arranged in the order of the molecules constituting the fibers, and their strength and physical properties are unlikely to be constant. Therefore, it is possible to impart appropriate strength and performance to the yarn by performing drawing. For example, when drawing a protein fiber, the higher the drawing ratio, the higher the drawing stress.
  • an object of the present invention is to provide a method for producing a protein fiber having excellent spinning stability and drawing stability.
  • the present invention provides the following [1] to [21].
  • [1] Introducing a spinning solution containing the protein, the first solvent, the second solvent and optionally a solubilizer into a coagulation bath solution containing a third solvent and optionally a solubilizer to coagulate the protein,
  • the method for producing a protein fiber wherein the second solvent is at least one selected from the group consisting of lower alcohols, ketones and water.
  • the third solvent is at least one selected from the group consisting of lower alcohols, ketones and water.
  • the first solvent is at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopropanol, hexafluoroacetone, formic acid, and water; However, the method according to [1] or [2], wherein when the first solvent is water, the spinning dope further contains a dissolution accelerator. [4] The method according to any one of [1] to [3], wherein the lower alcohol is methanol or ethanol. [5] The method according to any one of [1] to [4], wherein the protein is a structural protein. [6] The method according to [5], wherein the structural protein is fibroin.
  • a method for producing a protein fiber comprising a step of introducing a stock spinning solution containing a protein and a first solvent into a coagulation bath solution containing a second solvent to coagulate the protein, wherein the protein has an average particle diameter of 4%.
  • a method for producing a protein fiber which is dispersed in a stock spinning solution in a particle shape of 1515 nm.
  • the second solvent is at least one selected from the group consisting of lower alcohols, ketones and water.
  • the third solvent is at least one selected from the group consisting of lower alcohols, ketones and water.
  • the first solvent is at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopropanol, hexafluoroacetone, formic acid, and water;
  • the method according to any one of [13] to [15], wherein when the first solvent is water, the spinning dope further contains a dissolution accelerator.
  • the structural protein is fibroin.
  • the method for producing protein fibers of the present invention has excellent fiber-forming ability (yarn-forming properties) for spider silk proteins.
  • the protein fiber production method of the present invention can secure a fiber-forming ability (yarn-forming property), and increases the maximum draw ratio that can be wound between a nozzle and a roller, as compared with the conventional spinning method. It is also possible to adopt various yarn-making conditions. According to the method for producing a protein fiber of the present invention, a protein fiber having excellent stress can be provided. Further, since the draw ratio can be increased, thinner and longer protein fibers can be produced.
  • excellent spinning stability means that when an undrawn protein fiber is obtained from a spinning dope, yarn breakage is unlikely to occur.
  • excellent stretching stability means that a yarn is not easily broken when unstretched protein fiber is stretched.
  • the method for producing a protein fiber includes a step of introducing a stock spinning solution containing a protein, a first solvent, and a second solvent into a coagulation bath to coagulate the protein.
  • the spinning dope may optionally further contain a dissolution accelerator.
  • the coagulation bath solution contains a third solvent, and may optionally contain a dissolution accelerator.
  • the spinning solution used in the present embodiment contains a protein which is a main raw material of the protein fiber, a first solvent, and a second solvent.
  • 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.
  • the term “comprising as the 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.
  • the structural protein may be silk fibroin, spider silk fibroin or a combination thereof.
  • 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.
  • Silk fibroin may be sericin-removed silk fibroin, sericin-unremoved silk fibroin, 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 cord marker thread proteins, weft thread 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 spiders, and also has the characteristic of excellent toughness.
  • large spinal cord marker thread proteins include large ampullate gland spidroins MaSp1 and MaSp2 derived from the American spider (Nephila laclavipes), and ADF3 and ADF4 derived from 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.
  • a flagellated silk protein (flagelliform @ silk @ protein) derived from the American spider (Nephila @ clavipes) can be mentioned.
  • the artificial protein derived from the natural spider silk protein may be a recombinant spider silk protein.
  • 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.
  • 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.
  • A represents an alanine residue
  • 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.
  • 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.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • m represents an integer of 2 to 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.
  • 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.
  • 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.
  • 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).
  • PR1 sequence 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.
  • 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.
  • 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.
  • REP4 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 is 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.
  • 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: 13 (tag sequence) is located at the N-terminal of the amino acid sequence from the 19th residue to the 321st residue in the sequence obtained by replacing asparagine (Asn) at the 95th residue with aspartic acid (Asp). It has been added.
  • Proteins derived from elastin include, for example, NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine) and other proteins having an amino acid sequence.
  • 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).
  • Keratin-derived proteins include, for example, type I keratin of Capra hircus. 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).
  • 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.
  • 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, based on amino acid sequence information of structural proteins obtained from the NCBI web database or the like, AKTA ⁇ ⁇ ⁇ ⁇ oligopilotloplus 10/100 (GE Healthcare Japan)
  • the gene can be chemically synthesized by a method of linking the oligonucleotides automatically synthesized by the method such as PCR.
  • 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.
  • a plasmid vector a virus vector
  • a cosmid vector a fosmid vector
  • an artificial chromosome vector an artificial chromosome 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. .
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • microorganisms belonging to the genus Escherichia include, for example, Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri.
  • Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like.
  • 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.
  • examples of a vector into which a nucleic acid encoding a target protein is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold).
  • yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like.
  • filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
  • examples of a vector into which a nucleic acid encoding a desired protein is introduced include YEp13 (ATCC37115), YEp24 (ATCC37051), and the like.
  • 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.
  • a method using calcium ions [Proc. ⁇ Natl. ⁇ Acad. ⁇ Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culturing time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture 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.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium.
  • an inducer may be added to the medium as necessary.
  • lac promoter isopropyl- ⁇ -D-thiogalactopyranoside or the like is used.
  • 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 usually used for isolating and purifying a protein. 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.
  • 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).
  • a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei)
  • 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. Purification using methods such as alone or in combination It is possible to obtain the goods.
  • 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.
  • a purified sample of the protein can be obtained by the same isolation and purification method as described above.
  • 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.
  • the spinning solution can be prepared by mixing the protein, the first solvent, the second solvent and optionally a dissolution promoter. Specifically, it can be prepared by mixing a protein and a first solvent, optionally adding a dissolution promoter, and then adding a second solvent. If necessary, the mixture of the protein and the first solvent may be heated. The heating temperature can be adjusted according to the boiling point of the first solvent, for example, 35 to 90 ° C, 35 to 50 ° C, 35 to 45 ° C, 35 to 40 ° C, 50 to 90 ° C, 60 to 80 ° C. ° C, 80-90 ° C.
  • the addition of the second solvent can be adjusted according to the boiling point of the second solvent, for example, 10 to 90 ° C, 10 to 50 ° C, 50 to 90 ° C, 10 to 40 ° C, 60 to 80 ° C, 10 to It is preferable to carry out at 30 ° C., 15 to 30 ° C., or 20 to 30 ° C.
  • the protein exists in a state of being dissolved or dispersed in a mixed solvent of the first solvent and the second solvent in the spinning dope.
  • the protein fiber is obtained by spinning the above-mentioned protein, and contains the above-mentioned protein as a main component.
  • the content of the protein in the spinning dope may be 1 to 90% by mass based on the total weight of the spinning dope.
  • the protein content is 1% by mass or more, 2% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more, or 15% by mass or more based on the total mass of the spinning dope. It may be 40% by mass or less, 35% by mass or less, 30% by mass or less, or 25% by mass or less.
  • the first solvent may be any solvent capable of dissolving the protein, such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), hexafluoroisopronol (HFIP), hexafluoroacetone (HFAc), and formic acid (FAC). ) And water.
  • DMSO dimethyl sulfoxide
  • DMF N, N-dimethylformamide
  • HFIP hexafluoroisopronol
  • HFAc hexafluoroacetone
  • FAC formic acid
  • the first solvent may be used alone or in a combination of two or more.
  • the content of the first solvent in the spinning dope may be 50 to 90 parts by mass based on 100 parts by mass of the spinning dope.
  • the content of the first solvent may be 50 parts by mass or more, 60 parts by mass or more, 70 parts by mass or more, or 80 parts by mass or more with respect to 100 parts by mass of the spinning dope, and 90 parts by mass or less. It may be 80 parts by mass or less, 70 parts by mass or less, 60 parts by mass or less, 50 parts by mass or less.
  • the content of the first solvent is preferably 60 to 90 parts by mass, more preferably 60 to 85 parts by mass, still more preferably 65 to 85 parts by mass, and still more preferably 70 to 80 parts by mass with respect to 100 parts by mass of the spinning dope. Parts by weight, even more preferably 70 to 75 parts by weight.
  • the second solvent may be any solvent that can coagulate (desolventize) the protein by removing the first solvent from the spinning dope.
  • the second solvent may be, for example, at least one selected from the group consisting of lower alcohols, ketones, and water.
  • the second solvent may be used alone or in a combination of two or more.
  • the lower alcohol means a linear or branched alcohol having 1 to 5 carbon atoms, and specifically, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert- alcohol. Butanol, 1-pentanol, 2-pentanol, 3-pentanol, amyl alcohol, neopentyl alcohol and the like.
  • a preferred lower alcohol is methanol or ethanol, and a more preferred lower alcohol is ethanol.
  • Ketone is a compound represented by the formula (1): R 1 -C ((O) -R 2 .
  • R 1 and R 2 each independently represent a linear or branched alkyl group having 1 to 6 carbon atoms.
  • Specific examples of such a ketone include acetone, methyl ethyl ketone, methyl isobutyl ketone, and diisobutyl ketone.
  • R 1 and R 2 may form a ring structure with each other via a single bond.
  • Specific examples of such a cyclic ketone include cyclopentanone and cyclohexanone.
  • Preferred ketones are acetone or methyl ethyl ketone.
  • the content of the second solvent in the spinning dope may be 1 to 90 parts by mass based on 100 parts by mass of the protein to be dissolved. Further, the content of the second solvent is 1 part by mass or more, 5 parts by mass or more, 10 parts by mass or more, 20 parts by mass or more, 30 parts by mass or more, 40 parts by mass or more, based on 100 parts by mass of the protein to be dissolved.
  • the content of the second solvent is preferably 1 to 50 parts by mass, more preferably 1 to 40 parts by mass, still more preferably 5 to 40 parts by mass, and still more preferably 5 to 100 parts by mass with respect to 100 parts by mass of the protein to be dissolved. It is 30 parts by mass, still more preferably 5 to 20 parts by mass.
  • the stress in this specification means the value (unit: g / D) obtained by dividing the maximum load until the protein fiber is broken by the tensile external force in the fiber axis direction by the mass per 9000 m of the protein fiber.
  • the spinning dope may further contain a dissolution accelerator.
  • the spinning dope contains the dissolution promoter, the protein can be dissolved more easily and in a larger amount.
  • the spinning dope contains a dissolution accelerator.
  • the dissolution promoter can be appropriately selected according to the type of the protein and the dissolution solvent.
  • the dissolution promoter may be, for example, an inorganic salt composed of the following Lewis acid and Lewis base.
  • the Lewis base include oxo acid ions (nitrate ions, perchlorate ions, etc.), metal oxo acid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, and the like.
  • the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions.
  • examples of the inorganic salt include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, and calcium bromide.
  • Calcium salts such as calcium iodide, calcium nitrate, calcium perchlorate, and calcium thiocyanate; iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron thiocyanate; Aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate; potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and thiocyanate Potassium salts such as potassium acid, sodium chloride, nato bromide Sodium salts such as sodium, sodium iodide, sodium nitrate, sodium perchlorate and sodium thiocyanate; zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate and zinc thiocyanate Magnesium salts such as magnesium chloride,
  • inorganic salts are used as a promoter for dissolving a protein in a dissolution solvent.
  • the spinning dope contains a dissolution promoter (the above-mentioned inorganic salt)
  • the protein can be dissolved in the spinning dope at a high concentration.
  • the inorganic salt may be at least one selected from the group consisting of lithium chloride and calcium chloride.
  • the dissolution promoter may be used alone or in combination of two or more.
  • the content of the dissolution promoter is 0.1% by mass or more, 1% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more, or 15% by mass or more with respect to the total mass of the spinning dope. It may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less.
  • the spinning dope may further contain various additives as necessary.
  • the additives include a plasticizer, a leveling agent, a crosslinking agent, a crystal nucleating agent, an antioxidant, an ultraviolet absorber, a coloring agent, a filler, and a synthetic resin.
  • the content of the additive may be 50 parts by mass or less based on 100 parts by mass of the total amount of the protein in the spinning dope.
  • the method for producing a protein fiber according to the present embodiment includes a step of introducing the above-described spinning solution into a coagulation bath solution containing a third solvent and optionally a dissolution promoter to coagulate the protein (spinning step).
  • the method for producing a protein fiber according to the present embodiment can be performed according to a known spinning method such as wet spinning and dry-wet spinning.
  • the spinning solution is brought into contact with a coagulation bath solution to coagulate the protein.
  • the method for producing a protein fiber of the present embodiment, including the spinning step, can be performed using, for example, the spinning apparatus shown in FIG.
  • FIG. 1 is an explanatory view schematically showing an example of a spinning device for producing protein fibers.
  • the spinning device 10 shown in FIG. 1 is an example of a spinning device for dry-wet spinning, and includes an extrusion device 1, a coagulation bath 20, a washing bath (drawing bath) 21, and a drying device 4 in this order from the upstream side. are doing.
  • the extrusion device 1 has a storage tank 7 in which a stock spinning solution (dope solution) 6 is stored.
  • the coagulation bath liquid 11 is stored in the coagulation bath 20.
  • the spinning dope 6 is obtained by dissolving the protein described above in a dissolving solvent.
  • the stock spinning solution 6 is pushed out of a nozzle 9 provided with an air gap 19 opened between the stock solution 7 and a coagulation bath solution 11 by a gear pump 8 attached to the lower end of the storage tank 7.
  • the extruded spinning solution 6 is supplied (introduced) into the coagulation bath solution 11 of the coagulation bath tank 20 via the air gap 19.
  • the solvent is removed from the spinning dope to coagulate the protein.
  • the coagulated protein is guided to the washing bath 21 and washed by the washing liquid 12 in the washing bath 21, and then sent to the drying device 4 by the first nip roller 13 and the second nip roller 14 installed in the washing bath 21.
  • the protein fiber stretched in the washing liquid 12 is separated from the inside of the washing bath 21, dried when passing through the drying device 4, and then wound up by a winder. In this way, the protein fiber is finally obtained by the spinning device 10 as the wound material 5 wound on a winder.
  • 18a to 18g are yarn guides.
  • the coagulation bath solution 11 contains a third solvent.
  • the third solvent the solvent defined in the second solvent can be used.
  • the second solvent and the third solvent may be the same or different from each other.
  • the coagulation bath liquid 11 may appropriately contain water.
  • the content of the third solvent is preferably 60% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, based on the total mass of the coagulation bath liquid.
  • the content is even more preferably 90% by mass or more, and particularly preferably 95% by mass or more.
  • the coagulation bath liquid 11 may be composed of only the second solvent.
  • the content of water is preferably 30% by mass or less based on the total mass of the coagulation bath liquid.
  • the water content may be 20% by mass or less, or 10% by mass or less, based on the total mass of the coagulation bath liquid.
  • the coagulation bath liquid 11 may further contain a dissolution accelerator.
  • the dissolution promoter in the coagulation bath may be used alone or in combination of two or more.
  • the temperature of the coagulation bath solution 11 is not particularly limited, but may be 40 ° C or lower, 30 ° C or lower, 25 ° C or lower, 20 ° C or lower, 10 ° C or lower, or 5 ° C or lower.
  • the temperature of the coagulation bath liquid 11 is not particularly limited, but may be ⁇ 30 ° C. or higher, ⁇ 20 ° C. or higher, or ⁇ 10 ° C. or higher, and may be 0 ° C. or higher from the viewpoint of workability, cooling cost, and the like. preferable.
  • the temperature of the coagulation bath liquid 11 is within the above range, the generation of voids is sufficiently suppressed, the stress of the protein fiber is further increased, and the desired protein fiber is easily supplied stably.
  • the temperature of the coagulation bath liquid 11 can be adjusted by using, for example, a spinning device 10 including a coagulation bath 20 having a heat exchanger therein and a cooling circulation device.
  • a spinning device 10 including a coagulation bath 20 having a heat exchanger therein and a cooling circulation device.
  • a medium cooled to a predetermined temperature by a cooling circulation device through a heat exchanger installed in the coagulation bath 20, the temperature is adjusted to the above range by heat exchange between the coagulation bath liquid 11 and the heat exchanger. can do.
  • a solvent for example, methanol
  • a plurality of coagulation bath tanks 20 in which the coagulation bath liquid 11 is stored may be provided.
  • the coagulation bath liquid (first coagulation bath liquid) in the coagulation bath tank 20 to which the spinning dope 6 extruded from the nozzle 9 is directly supplied (introduced) may contain the third solvent. That is, when a plurality of coagulation baths 20 for storing the coagulation bath 11 are provided, even if the coagulation bath other than the first coagulation bath (other coagulation bath) does not contain the third solvent. Good.
  • the temperature of the other coagulation bath solution may be 40 ° C. or less, 30 ° C. or less, 25 ° C. or less, 20 ° C. or less, 10 ° C.
  • the temperature of the coagulation bath liquid is preferably 0 ° C. or higher.
  • the temperature of the coagulation bath liquid 11 can be adjusted by using, for example, a spinning device 10 including a coagulation bath 20 having a heat exchanger therein and a cooling circulation device. For example, by flowing a medium cooled to a predetermined temperature by a cooling circulation device through a heat exchanger installed in the coagulation bath 20, the temperature is adjusted to the above range by heat exchange between the coagulation bath liquid 11 and the heat exchanger. can do. In this case, more efficient cooling is possible by circulating the solvent used for the coagulation bath liquid 11 as a medium.
  • the coagulated protein may be taken up by a winder after leaving the coagulation bath or the washing bath, or may be passed through a drying device, dried, and then wound up by a winder.
  • the distance at which the coagulated protein passes through the coagulation bath solution 11 is such that the solvent can be efficiently removed and the extrusion speed of the spinning solution from the nozzle 9 is good. (Discharge speed) or the like may be determined.
  • the take-up speed of the undrawn yarn is, for example, 1 to 100 m / min, 1 to 20 m / min, and preferably 1 to 3 m / min. When the take-up speed is 1 m / min or more, the productivity can be sufficiently increased. When the take-up speed is 100 m / min or less, it is possible to avoid the scattering of the solvent liquid.
  • the residence time of the coagulated protein (or spinning solution) in the coagulation bath solution 11 is determined according to the distance that the coagulated protein passes through the coagulation bath solution 11, the extrusion speed of the spinning solution 6 from the nozzle 9, and the like. It may be.
  • the residence time may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulation bath liquid 11.
  • the method for producing a protein fiber of the present embodiment may further include a step of drawing the coagulated protein (drawing step).
  • the stretching step may be performed, for example, in the coagulation bath 20 or in the washing bath 21.
  • the stretching step can also be performed in air.
  • the stretching performed in the washing bath 21 may be so-called wet heat stretching performed in hot water, in a solution obtained by adding an organic solvent or the like to warm water, or the like.
  • the temperature for wet heat stretching is preferably from 50 to 90 ° C. When the temperature is 50 ° C. or higher, the pore diameter of the yarn can be stably reduced. When the temperature is 90 ° C. or lower, the temperature can be easily set and spinning stability is improved.
  • the temperature is more preferably from 75 to 85 ° C.
  • the final draw ratio of the undrawn yarn (or pre-drawn yarn) is preferably 5 times or more, 6 times or more, 7 times or more, 8 times or more, and 9 times or more of the undrawn yarn (or pre-drawn yarn).
  • the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less, 12 times or less, 11 times or less, and 10 times or less.
  • the undrawn yarn after drying and / or solvent removal may be drawn in water or may be drawn in two or more stages.
  • the underwater stretching is preferably performed at a water temperature of 20 to 90 ° C.
  • the drawn yarn is preferably heat-set at a dry heat of 50 to 200 ° C. for 5 to 600 seconds. By this heat setting, dimensional stability at normal temperature is obtained.
  • the drawing performed in the washing bath 21 when obtaining the protein fiber may be so-called wet heat drawing performed in hot water, a solution obtained by adding an organic solvent or the like to hot water, or the like.
  • the temperature for the wet heat stretching may be, for example, 50 to 90 ° C., preferably 75 to 85 ° C.
  • the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times.
  • the wet heat stretching and the dry heat stretching may be performed individually, or may be performed in multiple stages or in combination. That is, as the stretching step, the first-stage stretching is performed by wet-heat stretching, the second-stage stretching is performed by dry-heat stretching, or the first-stage stretching is performed by wet-heat stretching, the second-stage stretching is performed by wet-heat stretching, and the third-stage stretching is further performed.
  • wet heat stretching and dry heat stretching can be performed in an appropriate combination such as dry heat stretching.
  • the lower limit of the final draw ratio of the filament after the drawing step is preferably 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, or more with respect to the undrawn yarn (or pre-drawn yarn). It may be any of seven times, eight times, or nine times.
  • the upper limit of the final draw ratio of the multifilament after the drawing step is preferably any of 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. It may be.
  • the final stretching magnification may be 3 to 40 times, 3 to 30 times, 5 to 30 times, 5 to 20 times, or 5 to 15 times. And may be 5 to 13 times.
  • the present invention is a method for producing a protein fiber, comprising a step of introducing a spinning solution containing a protein and a first solvent into a coagulation bath solution containing a second solvent to coagulate the protein.
  • the present invention also has another aspect of a method for producing a protein fiber, wherein the average particle diameter of the protein contained in the stock solution is 4 to 15 nm.
  • the protein is dispersed with an average particle size of 4 to 15 nm.
  • the average particle size of the protein is preferably from 4 to 13 nm, more preferably from 4 to 10 nm, particularly preferably from 4 to 8 nm.
  • the stress of the protein fiber obtained by stretching after coagulation becomes higher, and the toughness is also improved.
  • ⁇ As a means for adjusting the average particle diameter of the protein to be within the above range, for example, a method of adding a specific amount of a second solvent to the spinning dope is exemplified.
  • the definitions of the first solvent and the second solvent are as described above.
  • the average particle size of the protein in the spinning stock solution is the average value of the particle size distribution.
  • the particle diameter is, for example, calculated based on an amount measured using a physical law derived when assuming a specific particle shape and specific physical conditions, Good.
  • the particle diameter and particle size distribution may be obtained by analyzing the fluctuation of the scattered light obtained when the particles are irradiated with light by the photon correlation method.
  • the average particle size of the protein is measured by dynamic light scattering using a dynamic light scattering photometer DLS-8000 (trade name, manufactured by Otsuka Electronics Co., Ltd.), and the average particle size based on the scattered light intensity is calculated. can do.
  • spider silk protein spike silk fibroin: PRT799
  • PRT799 modified fibroin having the amino acid sequence represented by SEQ ID NO: 12
  • the amino acid sequence represented by SEQ ID NO: 10 is obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes, and its N-terminal.
  • the amino acid sequence represented by SEQ ID NO: 11 amino acid sequence including a tag sequence and a hinge sequence
  • the amino acid sequence represented by SEQ ID NO: 12 (PRT799) is obtained by adding an amino acid sequence represented by SEQ ID NO: 6 (an amino acid sequence including a His tag sequence and a hinge sequence) to the N-terminal of the amino acid sequence represented by SEQ ID NO: 10. is there.
  • the designed 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). Thereafter, the nucleic acid was digested with NdeI and EcoRI and cut out, followed by recombination into a protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with the obtained pET22b (+) expression vector.
  • the transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 1) so that the OD 600 was 0.005.
  • the temperature of the culture was maintained at 30 ° C., and the flask was cultured for about 15 hours until the OD 600 reached 5, to obtain a seed culture.
  • the seed culture solution was added to a jar fermenter to which 500 ml of a production medium (Table 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.
  • the concentration of dissolved oxygen in the culture was maintained at 20% of the saturated concentration of dissolved oxygen.
  • a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) 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. Culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce PRT799 expression. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells. SDS-PAGE was performed using cells prepared from the culture solution before and after the addition of IPTG, and the expression of PRT799 was confirmed by the appearance of a band having a size corresponding to PRT799 depending on the addition of IPTG.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • 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 (PRT799) obtained after dialysis was recovered by centrifugation. The water was removed from the collected aggregated protein using a freeze dryer to obtain a freeze-dried powder of PRT799.
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • the average particle size of the protein (PRT799) dispersed in the obtained spinning stock solutions 1 to 3 was measured using a dynamic light scattering photometer DLS-8000 (trade name, manufactured by Otsuka Electronics Co., Ltd.). Table 7 shows the average particle diameter in each spinning dope.
  • Extrusion device 1: Extrusion device, 2: undrawn yarn production device, 3: wet heat drawing device, 4: drying device, 6: spinning solution, 10: spinning device, 20: coagulation bath, 21: washing bath, 36: protein fiber.

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  • Textile Engineering (AREA)
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Abstract

La présente invention concerne un procédé de production d'une fibre de protéine, le procédé comprenant une étape consistant à faire coaguler une protéine en introduisant une solution de filage, qui contient une protéine, un premier solvant, un deuxième solvant et éventuellement un promoteur de dissolution, dans un bain de coagulation qui contient un troisième solvant et éventuellement un promoteur de dissolution, le deuxième solvant étant au moins un élément choisi dans le groupe constitué par un alcool de faible poids moléculaire, une cétone et de l'eau.
PCT/JP2019/028547 2018-07-19 2019-07-19 Procédé de production de fibre de protéine WO2020017652A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06346314A (ja) * 1993-06-02 1994-12-20 Toyobo Co Ltd 再生絹フィブロイン繊維およびその製造方法
JPH07207520A (ja) * 1994-01-14 1995-08-08 Kiyoichi Matsumoto 絹フィブロイン繊維の製造法
JP2005163204A (ja) * 2003-12-01 2005-06-23 Univ Kansai ゼラチン繊維とその製造方法
WO2013065650A1 (fr) * 2011-11-02 2013-05-10 スパイバー株式会社 Solution polypeptidique, procédé de production d'une fibre polypeptidique synthétique, et procédé d'affinage de polypeptide
WO2013065651A1 (fr) * 2011-11-02 2013-05-10 スパイバー株式会社 Solution protéique et procédé de production d'une fibre protéique l'utilisant

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06346314A (ja) * 1993-06-02 1994-12-20 Toyobo Co Ltd 再生絹フィブロイン繊維およびその製造方法
JPH07207520A (ja) * 1994-01-14 1995-08-08 Kiyoichi Matsumoto 絹フィブロイン繊維の製造法
JP2005163204A (ja) * 2003-12-01 2005-06-23 Univ Kansai ゼラチン繊維とその製造方法
WO2013065650A1 (fr) * 2011-11-02 2013-05-10 スパイバー株式会社 Solution polypeptidique, procédé de production d'une fibre polypeptidique synthétique, et procédé d'affinage de polypeptide
WO2013065651A1 (fr) * 2011-11-02 2013-05-10 スパイバー株式会社 Solution protéique et procédé de production d'une fibre protéique l'utilisant

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