WO2021235417A1 - Polypeptide, nucleic acid, molded body, composition and method for producing same, and property improving agent - Google Patents

Polypeptide, nucleic acid, molded body, composition and method for producing same, and property improving agent Download PDF

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Publication number
WO2021235417A1
WO2021235417A1 PCT/JP2021/018695 JP2021018695W WO2021235417A1 WO 2021235417 A1 WO2021235417 A1 WO 2021235417A1 JP 2021018695 W JP2021018695 W JP 2021018695W WO 2021235417 A1 WO2021235417 A1 WO 2021235417A1
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Prior art keywords
polypeptide
composition
protein
film
nucleic acid
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PCT/JP2021/018695
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French (fr)
Japanese (ja)
Inventor
圭司 沼田
浩之 中村
和晴 荒川
暢明 河野
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Ｓｐｉｂｅｒ株式会社
学校法人慶應義塾
圭司 沼田
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Priority to JP2022502191A priority Critical patent/JP7177453B2/en
Publication of WO2021235417A1 publication Critical patent/WO2021235417A1/en

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    • 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
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

Definitions

  • the present invention relates to a polypeptide, a nucleic acid, a molded product, a composition and a method for producing the same, and an agent for improving physical properties.
  • Spider silk protein fibers containing artificial spider silk protein which have the characteristics of spider silk having excellent strength and high elasticity, have been studied for practical use (for example, Patent Document 1). It was
  • Patent Documents 2 and 3 disclose modified fibroin. It was
  • An object of the present invention is to provide a composition and a molded product having improved mechanical properties such as stress and elongation.
  • the present invention provides the following polypeptides, nucleic acids, molded products, compositions and methods for producing them, and physical property improving agents.
  • Molded product containing the polypeptide according to [1] [4] The molded product according to [3], wherein the molded product is a fiber or a film.
  • composition according to [5] wherein the content of the polypeptide is 1% or more with respect to the mass of the composition.
  • HFIP hexafluoroisopropanol
  • DMSO dimethyl sulfoxide
  • formic acid formic acid.
  • the composition according to [8], wherein the structural protein is fibroin.
  • the composition according to [9], wherein the fibroin is a spider silk protein.
  • a method for producing a composition comprising.
  • the method for producing a composition according to [12], wherein the composition is a protein molding solution.
  • the protein molding solution contains at least one solvent selected from the group consisting of HFIP, DMSO and formic acid.
  • the polypeptide provided in the present invention improves mechanical properties such as tensile strength when blended in a film. In addition, the elongation is improved when blended with fibers.
  • polypeptide of the present invention has 70% or more identity with the amino acid sequences 21 to 252 of SEQ ID NO: 1, and may be referred to as polypeptide I herein.
  • the polypeptide consisting of the 21st to 252nd amino acid sequences of SEQ ID NO: 1 is derived from Trichonephila clavata (Trichonephila clavata).
  • Polypeptide I has very low homology with known polypeptides, and no similar polypeptide has been found in spiders other than Nephila clavata. It was
  • the polypeptide of the invention may be tagged for purification.
  • Tags include His tag, glutathione-S-transferase (GST), maltose binding protein (MBP), ⁇ -galactosidase, digoxygenin (DIG), FITC, mini-AID, luciferase, GFP, RFP, thioredoxin (TRX), HA.
  • His tag glutathione-S-transferase (GST), maltose binding protein (MBP), ⁇ -galactosidase, digoxygenin (DIG), FITC, mini-AID, luciferase, GFP, RFP, thioredoxin (TRX), HA.
  • Tags myc tags, FLAG tags, V5 tags, S tags, E tags, T7 tags, VSV-G tags, Glu-Glu tags, Strep tags, HSV tags, chitin-binding domains (CBD), carmodulin-binding peptide
  • the polypeptide of the invention may contain a protease recognition sequence.
  • the protease recognition sequence include, but are not limited to, an HRV-3C recognition sequence, a sortase recognition sequence, a TEV protease recognition sequence, and a thrombin recognition sequence.
  • HRV-3C recognition sequence the recognition sequence LEVLFQ / GP (/: cleavage site) is cleaved by HRV-3C (protease).
  • the tag and the protease recognition sequence, the tag and the polypeptide I, and the protease recognition sequence and the polypeptide I may be directly bound, but can be bound via a linker.
  • the SSGSS sequence is used as a linker, but the present invention is not limited to this, and any linker including a known linker can be used. It was
  • the 6th Hiss from the 2nd to the 7th are His tags, and the SSGSSs from the 8th to the 12th are linker sequences.
  • the 13th to 18th LEVLFQ is a protease recognition sequence, and the 19th to 20th GP is a linker sequence.
  • the protease recognition sequence is cleaved between the 18th Gln and the 19th Gly of SEQ ID NO: 1, so that when cleaved with a protease, a polypeptide containing 234 amino acids from the 19th to the 252nd is produced. .. It was
  • the polypeptide of the present invention has an identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92 with the amino acid sequence from the 21st to the 252nd of SEQ ID NO: 1. % Or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100% identity, and film strength and / or transparency. Examples thereof include polypeptides having a function of improving the above.
  • the polypeptide of the present invention may further contain at least one selected from the group consisting of a protease recognition sequence, a linker, and a tag in addition to the above-mentioned 70% or more identity polypeptide. It was
  • the nucleic acid of the present invention encodes polypeptide I or is a complementary strand thereof.
  • the nucleic acids of the invention further include those encoding tags, linkers, protease recognition sequences. It was
  • composition of the present invention may contain polypeptide I, a solvent, and other macromolecules. It was
  • the solvent examples include water, N, N-dimethylformamide (DMF), dimethylacetamide, dimethylsulfoxide (DMSO), N-methylpyrrolidone, ethylene carbonate, propylene carbonate, dioxane, tetrahydrofuran (THF), formic acid, acetic acid and propionic acid.
  • alkylene glycol monolower alkyl ethers such as monomethyl ether
  • alkylene glycol dilower alkyl ethers such as ethylene glycol dimethyl ether
  • HFIP hexafluoroisopropanol
  • trifluoroacetic acid trifluoroacetic acid
  • trichloroacetic acid trifluoroethanol
  • other macromolecules means macromolecules other than polypeptide I.
  • Other macromolecules include structural proteins.
  • the structural protein include fibroin derived from spider silk or a variant thereof (for example, those exemplified in Patent Documents 2 and 3), fibroin derived from silk moth, collagen, elastin, keratin and resilin, and proteins derived from these. Can be done.
  • Polymers other than structural proteins include silk proteins such as sericin, casein, polyarginine, polyhistidine, polylysine, chitin, chitosan, gluten, protamine, gelatin, zein, soybean protein, ovoalbumin, polyhydroxybutyrate, etc.
  • Examples of the molded / artificial molded product containing the polypeptide I include fibers, films, sheets, non-woven fabrics, resins and the like. It was
  • the polypeptide I of the present invention can be mixed with the above-mentioned other polymers, solvents and the like and spun by a known spinning method to obtain fibers.
  • the spinning method prepares a composition containing the above-mentioned solvent and / or polymer and, if necessary, an additive such as a dissolution accelerator.
  • fibers can be obtained by spinning by a known spinning method such as wet spinning, dry spinning, dry wet spinning, and melt spinning.
  • Preferred spinning methods include wet spinning or dry-wet spinning. It was
  • the ratio of Polypeptide I contained in the composition such as fiber, film, sheet, non-woven fabric, resin, solution and the molded product is not particularly limited, but is 1% by mass or more, 3% by mass or more, 5% by mass or more, and 20% by mass. % Or less, 15% by mass or less, 10% by mass or less, 1 to 20% by mass, 1 to 15% by mass, 1 to 10% by mass, 3 to 20% by mass, 3 to 15% by mass, 3 to 10% by mass, 5 It is ⁇ 20% by mass, 5 ⁇ 15% by mass, and 5 ⁇ 10% by mass. It was
  • the mechanical properties of the artificial composition can be improved, and when the composition and the molded product are films, the permeability is improved.
  • An artificial composition and an artificial molded body that can be made to be provided are provided. That is, the artificial composition and the artificial molded product according to one embodiment contain another polymer and polypeptide I.
  • the "artificial composition” and “artificial molded body” are, for example, a composition and a molded body containing a protein produced by a microorganism or the like by a genetic recombination technique or another polymer produced by a chemical synthesis. means. It was
  • the artificial composition and the artificial molded product may be the above-mentioned structural proteins, and the preferred structural protein aspects are the same as those described above.
  • the artificial composition and the artificial molded product may be, for example, fibers, films, sheets, non-woven fabrics, resins, solutions and the like. It was
  • the polypeptide I of the present invention is formed into a film by dissolving or dispersing in a solvent together with the polymer as needed, casting the obtained solution or dispersion on the surface of a substrate, drying and removing the solvent. can do. It was
  • the base material may be a resin substrate, a glass substrate, a metal substrate, or the like.
  • the base material is preferably a resin substrate from the viewpoint that the film after cast molding can be easily peeled off.
  • the resin substrate may be, for example, a polyethylene terephthalate (PET) film, a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a release film in which a silicone compound is immobilized on the surface of these films.
  • PET polyethylene terephthalate
  • PP polypropylene
  • the silicone compound is more preferably fixed on the PET film or the surface of the PET film. It is a made release film. It was
  • Drying and / or desolvation is performed by at least one means selected from, for example, vacuum drying, hot air drying, air drying, and immersion in liquid.
  • the immersion in the liquid may be an alcohol solution such as water, a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol, or 2-propanol, or a cast film may be immersed in a mixed solution of water and alcohol to remove the solvent. good.
  • the temperature of the desolvent solution (coagulant solution) is preferably 0 to 90 ° C. It is preferable to remove the solvent as much as possible.
  • the unstretched film after drying and / or desolving can be uniaxially stretched or biaxially stretched in water.
  • the biaxial stretching may be sequential stretching or simultaneous biaxial stretching. Two or more stages of multi-stage stretching may be performed.
  • the draw ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times in both the vertical and horizontal directions. Within this range, the stress-strain balance is easy to achieve.
  • the underwater stretching is preferably performed at a water temperature of 20 to 90 ° C.
  • the stretched film is preferably heat-fixed at 50 to 200 ° C. for 5 to 600 seconds. By this heat fixing, dimensional stability at room temperature can be obtained.
  • the uniaxially stretched film is a uniaxially oriented film
  • the biaxially stretched film is a biaxially oriented film. It was
  • the film may be a color film.
  • a colorant such as a dye is dissolved or dispersed in a DMSO solvent, for example, to prepare a DMSO coloring liquid, and the solution obtained by mixing the coloring liquid and the doping liquid is cast-molded in the same manner as described above.
  • a film is dried and / or desolvated to obtain an unstretched colored film, or stretched to obtain a stretched film.
  • the color film can be applied to a reflector, a marker, an ultraviolet protection film, a slit thread, and the like. It was
  • the thickness of the film can be adjusted according to the intended use and the like, and is, for example, 1 to 100 ⁇ m. It was
  • the polypeptide I of the present invention can be dissolved or dispersed in a solvent together with other polymers as needed to form a solution or dispersion, and the solution or dispersion can be made into a nonwoven fabric by papermaking or the like.
  • a composition containing polypeptide I, an adhesive, a solvent, another polymer, etc. is used as a raw material, and a dry method, a wet method, a spunbond method, a melt blown method, a thermal bond method, or a chemical bond method (impregnation method, spray method) is used.
  • Needle punching method, water flow entanglement method, etc. can also be used to form the non-woven fabric. It was
  • the thickness of the nonwoven fabric can be adjusted according to the intended use and the like, and may be, for example, 0.1 ⁇ m to 2 mm or 0.1 ⁇ m to 500 ⁇ m. It is preferably 1 to 100 ⁇ m.
  • Reference Example 1 (1) Preparation of plasmid expression strain
  • the spider silk protein MaSp2 was designed based on the base sequence and amino acid sequence of fibroin derived from Trichonephila clavata (Trichonephila clavata).
  • the base sequence encoding MaSp2 encodes an amino acid sequence (SEQ ID NO: 3) in which amino acid residues have been substituted, inserted or deleted for the purpose of improving productivity.
  • SEQ ID NO: 3 the 6th Hiss from the 2nd to the 7th are His tags, and the SSGSSs from the 8th to the 12th are linker sequences.
  • the 13th to 18th LEVLFQ is a protease recognition sequence
  • the 19th to 20th GP is a linker sequence
  • the 21st to 540th is a MaSp2 coding region.
  • the protease recognition sequence is cleaved between 18th Gln and 19th Gly of SEQ ID NO: 3, so when cleaved with protease, a polypeptide containing 522 amino acids 19th to 540th is produced. .. It was
  • nucleic acid encoding MaSp2 was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector. It was
  • the seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 2) was added so that the OD was 0.05.
  • the temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration. It was
  • the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of MaSp2. Twenty hours after the addition of IPTG, the culture broth was centrifuged and the cells were collected.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity.
  • the washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 30 at 60 ° C. Stir for minutes with a stirrer to dissolve.
  • 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 dialysis was recovered by centrifugation, water was removed by a freeze-dryer, and the freeze-dried powder was recovered to obtain spider silk protein MaSp2.
  • Example 1 Protein synthesis (Polypeptide I) Transcriptome analysis using the predecessor of Trichonephila clavata (Joro spider) and the silk gland revealed a relatively highly expressed transcript at the end of the silk gland. Based on the base sequence and amino acid sequence of the protein of unknown function encoded by this transcript, a spider silk protein having the amino acid sequence shown by SEQ ID NO: 1 was designed. The amino acid sequence shown by SEQ ID NO: 1 has a signal sequence removed from the amino acid sequence of a protein of unknown function derived from Joro spider, and has a His tag sequence (HHHHHH), a linker sequence (SSGSS), and a protease recognition site at the N-terminal. (LEVLFQGP) is added. It was
  • nucleic acid sequence encoding polypeptide I (SEQ ID NO: 2) was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector. It was
  • the seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 4) was added so that the OD was 0.05.
  • the temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration. It was
  • the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of polypeptide I.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • Ni-NTA manufactured by Biorad
  • Ni-NTA is thoroughly washed with sodium phosphate buffer (pH 7) containing 15 mM imidazole, 1 mM DTT, and 7.5 M urea, and then 500 mM imidazole.
  • Polypeptide I was eluted and recovered using sodium phosphate buffer (pH 7) containing 1 mM DTT and 7.5 M urea.
  • the fraction containing the obtained polypeptide I was dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 7.5 M urea, 1 mM DTT, and 50 mM sodium chloride, and the buffer was replaced.
  • the centrifuge tube After heating to dissolve the spider silk protein, the centrifuge tube was opened, and the protein solution was developed on a petri dish and allowed to stand at room temperature for 16 hours to dry to obtain a spider silk protein film. It was
  • the spider silk protein film was cut into a rectangular shape and used as a test piece (5 mm ⁇ 15 mm).
  • a tensile test was carried out using a test piece using a tensile test device EZ-LX HS manufactured by Shimadzu Corporation.
  • the fracture point displacement (strain) (%), maximum point stress (MPa), Young's modulus (MPa), and toughness ([MJ] / [m 3 ]) were measured by a tensile test.
  • spider silk protein fiber 1.66 g of spider silk protein MaSp2 was added to DMSO 5.89 g prepared so that LiCl was 4% by mass, and heated and stirred at 90 ° C for 8 hours using a temperature controller, and the protein concentration was 22 mass. A percentage protein solution was obtained. It was
  • a known dry-wet spinning was performed using the doping solution obtained as described above and the in-house spinning apparatus shown in FIG. 1, and spider silk protein fibers were wound around a bobbin.
  • dry-wet spinning was performed under the following conditions. Coagulant (methanol) temperature: 5 to 10 ° C Stretching ratio: 5.5 times, 6.0 times Drying temperature: 70 ° C
  • Table 9 shows the values of tensile strength, fracture point displacement, Young's modulus, fiber diameter, and centrifugal magnification of each sample obtained from the tensile test results. It was

Abstract

[Problem] To provide a composition and a molded body having improved mechanical properties such as stress and elongation. [Solution] A polypeptide that comprises an amino acid sequence having 70% or higher identity to the amino acid sequence consisting of from the 21st to 252nd amino acids in SEQ ID NO: 1.

Description

ポリペプチド、核酸、成形体、組成物及びその製造方法、並びに物性向上剤Polypeptides, nucleic acids, molded articles, compositions and methods for producing them, and physical property improving agents.
本発明は、ポリペプチド、核酸、成形体、組成物及びその製造方法、並びに物性向上剤に関する。 The present invention relates to a polypeptide, a nucleic acid, a molded product, a composition and a method for producing the same, and an agent for improving physical properties.
優れた強度及び高い伸縮性を有するクモ糸の特徴を備えた、人工クモ糸タンパク質を含むクモ糸タンパク質繊維は実用化に向けて研究されている（例えば、特許文献１）。　 Spider silk protein fibers containing artificial spider silk protein, which have the characteristics of spider silk having excellent strength and high elasticity, have been studied for practical use (for example, Patent Document 1). It was
特許文献２、３は、改変フィブロインを開示している。　 Patent Documents 2 and 3 disclose modified fibroin. It was
タンパク質を工業用材料して利用する場合、製品として要求される性能に耐えうる性能を有していること必要とされる。 When a protein is used as an industrial material, it is required to have a performance that can withstand the performance required as a product.
特許第５５４０１５４号公報Japanese Patent No. 5540154 WO2019/194224WO2019 / 194224 WO2019/022163WO2019 / 022163
本発明は、応力、伸度などの機械的物性を向上させた組成物及び成形体を提供することを目的とする。 An object of the present invention is to provide a composition and a molded product having improved mechanical properties such as stress and elongation.
本発明は、以下のポリペプチド、核酸、成形体、組成物及びその製造方法、並びに物性向上剤を提供するものである。〔１〕　配列番号１の21番目から252番目のアミノ酸配列と７０％以上の同一性を有するポリペプチド。〔２〕　〔１〕のポリペプチドをコードする核酸またはその相補鎖。〔３〕　〔１〕に記載のポリペプチドを含む成形体〔４〕　前記成形体が繊維又はフィルムである、〔３〕に記載の成形体。〔５〕　〔１〕に記載のポリペプチドと、溶媒及び他の高分子からなる群から選ばれる少なくとも１種を含む、組成物。〔６〕　前記ポリペプチドの含有量が前記組成物の質量に対して1％以上である、〔５〕に記載の組成物。〔７〕　前記組成物がHFIP(ヘキサフルオロイソプロパノール)、DMSO（ジメチルスルホキシド）及びギ酸からなる群から選ばれる少なくとも１種の溶媒を含む、〔５〕又は〔６〕に記載の組成物。〔８〕　前記他の高分子が構造タンパク質である〔５〕～〔７〕のいずれか１項に記載の組成物。〔９〕　前記構造タンパク質がフィブロインである、〔８〕に記載の組成物。〔１０〕　前記フィブロインがクモ糸タンパク質である、〔９〕に記載の組成物。〔１１〕　〔１〕のポリペプチドを含む、成形体の物性向上剤。〔１２〕　配列番号１の21番目から252番目のアミノ酸配列と７０％以上の同一性を有するポリペプチド含む組成物を製造する方法であって、前記ポリペプチドと他の高分子とを混合する工程を含む組成物の製造方法。〔１３〕　前記組成物が、タンパク質成形用溶液である〔１２〕に記載の組成物の製造方法。〔１４〕　前記タンパク質成形用溶液がHFIP、DMSO及びギ酸からなる群から選ばれる少なくとも１種の溶媒を含む、〔１３〕に記載の組成物の製造方法。〔１５〕　他の高分子が構造タンパク質である〔１２〕～〔１４〕のいずれか１項に記載の組成物の製造方法。〔１６〕　前記構造タンパク質がフィブロインである〔１５〕に記載の組成物の製造方法。〔１７〕　前記フィブロインがクモ糸タンパク質である〔１６〕に記載の組成物の製造方法。〔１８〕　前記組成物がフィルム又は繊維である〔１２〕～〔１７〕のいずれか１項に記載の組成物の製造方法。 The present invention provides the following polypeptides, nucleic acids, molded products, compositions and methods for producing them, and physical property improving agents. [1] A polypeptide having 70% or more identity with the 21st to 252nd amino acid sequences of SEQ ID NO: 1. [2] Nucleic acid encoding the polypeptide of [1] or a complementary strand thereof. [3] Molded product containing the polypeptide according to [1] [4] The molded product according to [3], wherein the molded product is a fiber or a film. [5] A composition containing the polypeptide according to [1] and at least one selected from the group consisting of a solvent and other polymers. [6] The composition according to [5], wherein the content of the polypeptide is 1% or more with respect to the mass of the composition. [7] The composition according to [5] or [6], wherein the composition contains at least one solvent selected from the group consisting of HFIP (hexafluoroisopropanol), DMSO (dimethyl sulfoxide) and formic acid. [8] The composition according to any one of [5] to [7], wherein the other polymer is a structural protein. [9] The composition according to [8], wherein the structural protein is fibroin. [10] The composition according to [9], wherein the fibroin is a spider silk protein. [11] An agent for improving the physical properties of a molded product, which contains the polypeptide of [1]. [12] A method for producing a composition containing a polypeptide having 70% or more identity with the 21st to 252nd amino acid sequences of SEQ ID NO: 1, a step of mixing the polypeptide with another polymer. A method for producing a composition comprising. [13] The method for producing a composition according to [12], wherein the composition is a protein molding solution. [14] The method for producing a composition according to [13], wherein the protein molding solution contains at least one solvent selected from the group consisting of HFIP, DMSO and formic acid. [15] The method for producing a composition according to any one of [12] to [14], wherein the other polymer is a structural protein. [16] The method for producing a composition according to [15], wherein the structural protein is fibroin. [17] The method for producing a composition according to [16], wherein the fibroin is a spider silk protein. [18] The method for producing a composition according to any one of [12] to [17], wherein the composition is a film or a fiber.
本発明で提供されるポリペプチドは、フィルムに配合した場合に引張強度などの機械的特性が向上する。また、繊維に配合した場合に伸度が向上する。 The polypeptide provided in the present invention improves mechanical properties such as tensile strength when blended in a film. In addition, the elongation is improved when blended with fibers.
ポリペプチドＩを含む繊維の製造装置Equipment for producing fibers containing polypeptide I
本発明のポリペプチドは、配列番号１の21番目から252番目のアミノ酸配列と７０％以上の同一性を有するものであり、本明細書においてポリペプチドＩと記載することがある。配列番号１の21番目から252番目のアミノ酸配列からなるポリペプチドは、Trichonephila clavata（ジョロウグモ）に由来する。ポリペプチドＩは、公知のポリペプチドとの相同性は非常に低く、ジョロウグモ上科以外の蜘蛛には類似のポリペプチドは見出されていない。　 The polypeptide of the present invention has 70% or more identity with the amino acid sequences 21 to 252 of SEQ ID NO: 1, and may be referred to as polypeptide I herein. The polypeptide consisting of the 21st to 252nd amino acid sequences of SEQ ID NO: 1 is derived from Trichonephila clavata (Trichonephila clavata). Polypeptide I has very low homology with known polypeptides, and no similar polypeptide has been found in spiders other than Nephila clavata. It was
本発明のポリペプチドは精製のためのタグを結合してもよい。タグとしては、Hisタグ、グルタチオン-S-トランスフェラーゼ（GST）、マルトース結合タンパク質（MBP）、β－ガラクトシダーゼ、ジゴキシゲニン(DIG)、FITC、mini-AID、ルシフェラーゼ、GFP、RFP、チオレドキシン(TRX)、HAタグ、mycタグ、FLAGタグ、V5タグ、Sタグ、Eタグ、T7タグ、VSV-Gタグ、Glu-Gluタグ、Strepタグ、HSVタグ、キチン結合ドメイン(CBD)、カルモジュリン結合ペプチド(CBP)などが挙げられる。　 The polypeptide of the invention may be tagged for purification. Tags include His tag, glutathione-S-transferase (GST), maltose binding protein (MBP), β-galactosidase, digoxygenin (DIG), FITC, mini-AID, luciferase, GFP, RFP, thioredoxin (TRX), HA. Tags, myc tags, FLAG tags, V5 tags, S tags, E tags, T7 tags, VSV-G tags, Glu-Glu tags, Strep tags, HSV tags, chitin-binding domains (CBD), carmodulin-binding peptides (CBP), etc. Can be mentioned. It was
本発明のポリペプチドは、プロテアーゼ認識配列を含んでいてもよい。プロテアーゼ認識配列としては、例えばHRV-3C認識配列、ソルターゼ認識配列、TEVプロテアーゼ認識配列、トロンビン認識配列等が挙げられるが、これらに限定されない。例えばHRV-3C認識配列は、HRV-3C（プロテアーゼ）により、その認識配列LEVLFQ／GP（／：切断部位）が切断される。　 The polypeptide of the invention may contain a protease recognition sequence. Examples of the protease recognition sequence include, but are not limited to, an HRV-3C recognition sequence, a sortase recognition sequence, a TEV protease recognition sequence, and a thrombin recognition sequence. For example, in the HRV-3C recognition sequence, the recognition sequence LEVLFQ / GP (/: cleavage site) is cleaved by HRV-3C (protease). It was
タグとプロテアーゼ認識配列、タグとポリペプチドＩ、プロテアーゼ認識配列とポリペプチドＩは、直接結合してもよいが、リンカーを介して結合することができる。本願実施例ではSSGSS配列がリンカーとして使用されているが、これに限定されることはなく、公知のリンカーを含む任意のリンカーを使用することができる。　 The tag and the protease recognition sequence, the tag and the polypeptide I, and the protease recognition sequence and the polypeptide I may be directly bound, but can be bound via a linker. In the examples of the present application, the SSGSS sequence is used as a linker, but the present invention is not limited to this, and any linker including a known linker can be used. It was
配列番号１のポリペプチドにおいて、２番目から７番目の６個のHisはHisタグであり、８番目から１２番目のSSGSSはリンカー配列である。また、１３番目から１８番目のLEVLFQはプロテアーゼ認識配列であり、１９番目から２０番目のGPはリンカー配列である。プロテアーゼ認識配列は、配列番号１の18番目のGlnと19番目のGlyの間で切断されるので、プロテアーゼで切断されたときに19番目から252番目の234個のアミノ酸を含むポリペプチドが生成する。　 In the polypeptide of SEQ ID NO: 1, the 6th Hiss from the 2nd to the 7th are His tags, and the SSGSSs from the 8th to the 12th are linker sequences. The 13th to 18th LEVLFQ is a protease recognition sequence, and the 19th to 20th GP is a linker sequence. The protease recognition sequence is cleaved between the 18th Gln and the 19th Gly of SEQ ID NO: 1, so that when cleaved with a protease, a polypeptide containing 234 amino acids from the 19th to the 252nd is produced. .. It was
本発明のポリペプチドは、配列番号１の21番目から252番目までのアミノ酸配列との同一性が７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、９１％以上、９２％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上又は１００％の同一性を有し、かつ、フィルムの強度及び/又は透明性を向上させる機能を有するポリペプチドが挙げられる。本発明のポリペプチドは、上記の７０％以上の同一性のポリペプチドに、さらにプロテアーゼ認識配列、リンカー、タグからなる群から選ばれる少なくとも１種を含んでいてもよい。　 The polypeptide of the present invention has an identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92 with the amino acid sequence from the 21st to the 252nd of SEQ ID NO: 1. % Or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100% identity, and film strength and / or transparency. Examples thereof include polypeptides having a function of improving the above. The polypeptide of the present invention may further contain at least one selected from the group consisting of a protease recognition sequence, a linker, and a tag in addition to the above-mentioned 70% or more identity polypeptide. It was
本発明の核酸は、ポリペプチドＩをコードするもの或いはその相補鎖である。本発明の核酸は、さらにタグ、リンカー、プロテアーゼ認識配列をコードするものも包含する。　 The nucleic acid of the present invention encodes polypeptide I or is a complementary strand thereof. The nucleic acids of the invention further include those encoding tags, linkers, protease recognition sequences. It was
本発明の組成物、特に人工組成物は、ポリペプチドＩと溶媒、他の高分子を含んでいてもよい。　 The composition of the present invention, particularly an artificial composition, may contain polypeptide I, a solvent, and other macromolecules. It was
前記溶媒としては、水、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、ジメチルアセトアミド、ジメチルスルホキシド（ＤＭＳＯ）、Ｎ－メチルピロリドン、炭酸エチレン、炭酸プロピレン、ジオキサン、テトラヒドロフラン（ＴＨＦ）、ギ酸、酢酸、プロピオン酸などの炭素数１～６、好ましくは１～４のモノカルボン酸、メタノール、エタノール、n-プロパノール、イソプロパノール、n-ブタノール等の低級アルコール、エチレングリコール、プロピレングリコール等のグリコール類、グリセリン、エチレングリコールモノメチルエーテル等のアルキレングリコールモノ低級アルキルエーテル、エチレングリコールジメチルエーテル等のアルキレングリコールジ低級アルキルエーテル、ヘキサフルオロイソプロパノール（ＨＦＩＰ）、トリフルオロ酢酸、トリクロロ酢酸、パーフルオロエタノールなどが挙げられる。　 Examples of the solvent include water, N, N-dimethylformamide (DMF), dimethylacetamide, dimethylsulfoxide (DMSO), N-methylpyrrolidone, ethylene carbonate, propylene carbonate, dioxane, tetrahydrofuran (THF), formic acid, acetic acid and propionic acid. Monocarboxylic acids having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, lower alcohols such as methanol, ethanol, n-propanol, isopropanol and n-butanol, glycols such as ethylene glycol and propylene glycol, glycerin and ethylene glycol. Examples thereof include alkylene glycol monolower alkyl ethers such as monomethyl ether, alkylene glycol dilower alkyl ethers such as ethylene glycol dimethyl ether, hexafluoroisopropanol (HFIP), trifluoroacetic acid, trichloroacetic acid, and perfluoroethanol. It was
本明細書において、「他の高分子」はポリペプチドＩ以外の高分子を意味する。他の高分子としては、構造タンパク質が含まれる。構造タンパク質としては蜘蛛糸由来のフィブロイン又はその改変体(例えば特許文献２，３に例示されるもの)、カイコ由来のフィブロイン、コラーゲン、エラスチン、ケラチン及びレシリン、並びにこれらに由来するタンパク質等を挙げることができる。構造タンパク質以外の他の高分子としては、セリシンなどのシルクプロテイン、カゼイン、ポリアルギニン、ポリヒスチジン、ポリリジン、キチン、キトサン、グルテン、プロタミン、ゼラチン、ゼイン、大豆タンパク、オボアルブミン、ポリヒドロキシブチレート、ポリカプロラクトン、ポリブチレンサクシネート、ポリブチレンサクシネート／アジペート、ポリエチレンサクシネート、ポリ乳酸、ポリグリコール酸、乳酸－グリコール酸共重合体、デンプン、セルロース、メチルセルロース、エチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルメチルセルロースアセテートサクシネート、ポリビニルピロリドン、カルボキシメチルスターチナトリウム、ポリエステル、ポリプロピレン、ポリエチレン、ポリアミド、ポリカーボネート、再生セルロース、酢酸セルロース、セルロースナノファイバー、シルクナノファイバーなどのナノファイバー、接着剤などが挙げられる。　 As used herein, "other macromolecules" means macromolecules other than polypeptide I. Other macromolecules include structural proteins. Examples of the structural protein include fibroin derived from spider silk or a variant thereof (for example, those exemplified in Patent Documents 2 and 3), fibroin derived from silk moth, collagen, elastin, keratin and resilin, and proteins derived from these. Can be done. Polymers other than structural proteins include silk proteins such as sericin, casein, polyarginine, polyhistidine, polylysine, chitin, chitosan, gluten, protamine, gelatin, zein, soybean protein, ovoalbumin, polyhydroxybutyrate, etc. Polycaprolactone, Polybutylene Succinate, Polybutylene Succinate / Adipate, Polyethylene Succinate, Polylactic Acid, Polyglycolic Acid, Lactic Acid-Glycolic Acid Polymer, Starch, Cellulose, Methyl Cellulose, Ethyl Cellulose, Hydroxyethyl Cellulose, Hydroxypropyl Methyl Cellulose Acetate Succi Examples thereof include nanofibers such as nate, polyvinylpyrrolidone, sodium carboxymethyl starch, polyester, polypropylene, polyethylene, polyamide, polycarbonate, regenerated cellulose, cellulose acetate, cellulose nanofibers and silk nanofibers, and adhesives. It was
ポリペプチドＩを含む成形体／人工成形体としては、繊維、フィルム、シート、不織布、樹脂などが挙げられる。　 Examples of the molded / artificial molded product containing the polypeptide I include fibers, films, sheets, non-woven fabrics, resins and the like. It was
本発明のポリペプチドＩは、上記の他の高分子、溶媒などと混合して公知の紡糸方法で紡糸することにより繊維を得ることができる。紡糸方法は、具体的には、上記の溶媒及び／又は高分子など、必要に応じてさらに溶解促進剤などの添加剤を含む組成物を作製する。次いで、この組成物を用いて、湿式紡糸、乾式紡糸、乾湿式紡糸、溶融紡糸等の公知の紡糸方法により紡糸して、繊維を得ることができる。好ましい紡糸方法としては、湿式紡糸又は乾湿式紡糸を挙げることができる。　 The polypeptide I of the present invention can be mixed with the above-mentioned other polymers, solvents and the like and spun by a known spinning method to obtain fibers. Specifically, the spinning method prepares a composition containing the above-mentioned solvent and / or polymer and, if necessary, an additive such as a dissolution accelerator. Then, using this composition, fibers can be obtained by spinning by a known spinning method such as wet spinning, dry spinning, dry wet spinning, and melt spinning. Preferred spinning methods include wet spinning or dry-wet spinning. It was
繊維、フィルム、シート、不織布、樹脂、溶液などの組成物及び成形体に含まれるポリペプチドＩの割合は、特に限定されないが、1質量％以上、3質量％以上、5質量％以上、20質量％以下、15質量％以下、10質量％以下、1～20質量％、1～15質量％、1～10質量％、3～20質量％、3～15質量％、3～10質量％、5～20質量％、5～15質量％、5～10質量％である。　 The ratio of Polypeptide I contained in the composition such as fiber, film, sheet, non-woven fabric, resin, solution and the molded product is not particularly limited, but is 1% by mass or more, 3% by mass or more, 5% by mass or more, and 20% by mass. % Or less, 15% by mass or less, 10% by mass or less, 1 to 20% by mass, 1 to 15% by mass, 1 to 10% by mass, 3 to 20% by mass, 3 to 15% by mass, 3 to 10% by mass, 5 It is ~ 20% by mass, 5 ~ 15% by mass, and 5 ~ 10% by mass. It was
このようなポリペプチドＩの特性を利用して、一実施形態においては、人工組成物の機械的物性を向上させることができ、かつ、組成物及び成形体がフィルムである場合は透過性を向上させることができる人工組成物、人工成形体が提供される。すなわち、一実施形態に係る人工組成物、人工成形体は、他の高分子とポリペプチドＩとを含む。なお、「人工組成物」、「人工成形体」とは、例えば、遺伝子組換え技術により微生物等で製造したタンパク質や、化学合成により製造された他の高分子を含む組成物及び成形体等を意味する。　 Utilizing such characteristics of Polypeptide I, in one embodiment, the mechanical properties of the artificial composition can be improved, and when the composition and the molded product are films, the permeability is improved. An artificial composition and an artificial molded body that can be made to be provided are provided. That is, the artificial composition and the artificial molded product according to one embodiment contain another polymer and polypeptide I. The "artificial composition" and "artificial molded body" are, for example, a composition and a molded body containing a protein produced by a microorganism or the like by a genetic recombination technique or another polymer produced by a chemical synthesis. means. It was
人工組成物及び人工成形体は上述した構造タンパク質であってよく、好ましい構造タンパク質の態様も上述したのと同様である。人工組成物及び人工成形体は、例えば、繊維、フィルム、シート、不織布、樹脂、溶液等であってよい。　 The artificial composition and the artificial molded product may be the above-mentioned structural proteins, and the preferred structural protein aspects are the same as those described above. The artificial composition and the artificial molded product may be, for example, fibers, films, sheets, non-woven fabrics, resins, solutions and the like. It was
本発明のポリペプチドＩは、必要に応じて前記高分子とともに溶媒に溶解又は分散し、得られた溶液又は分散液を基材表面にキャストし、乾燥及び脱溶媒を行うことで、フィルムに成形することができる。　 The polypeptide I of the present invention is formed into a film by dissolving or dispersing in a solvent together with the polymer as needed, casting the obtained solution or dispersion on the surface of a substrate, drying and removing the solvent. can do. It was
基材は、樹脂基板、ガラス基板、金属基板等であってよい。基材は、キャスト成形後のフィルムを容易に剥離できる観点から、好ましくは樹脂基板である。樹脂基板としては、例えば、ポリエチレンテレフタレート（ＰＥＴ）フィルム、ポリテトラフルオロエチレン等のフッ素樹脂フィルム、ポリプロピレン（ＰＰ）フィルム、又はこれらのフィルム表面にシリコーン化合物を固定化させた剥離フィルムであってよい。基材は、ＤＭＳＯ溶媒に対して安定であり、ドープ溶液を安定してキャスト成形でき、成形後のフィルムを容易に剥離できる観点から、より好ましくは、ＰＥＴフィルム又はＰＥＴフィルム表面にシリコーン化合物を固定化させた剥離フィルムである。　 The base material may be a resin substrate, a glass substrate, a metal substrate, or the like. The base material is preferably a resin substrate from the viewpoint that the film after cast molding can be easily peeled off. The resin substrate may be, for example, a polyethylene terephthalate (PET) film, a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a release film in which a silicone compound is immobilized on the surface of these films. From the viewpoint that the base material is stable to the DMSO solvent, the dope solution can be stably cast-molded, and the film after molding can be easily peeled off, the silicone compound is more preferably fixed on the PET film or the surface of the PET film. It is a made release film. It was
乾燥及び／又は脱溶媒は、例えば、真空乾燥、熱風乾燥、風乾、及び
液中浸漬から選ばれる少なくとも一種の手段で行われる。液中浸漬は、水中、メタノール、エタノール、２－プロパノールなどの炭素数１～５の低級アルコールなどのアルコール液でもよいし、水とアルコール混合液などにキャストフィルムを浸漬して脱溶媒させてもよい。脱溶媒液（凝固液）の温度は、好ましくは０～９０℃である。溶媒はできるだけ脱離したほうが好ましい。なお、フィルムを液中で延伸する場合、脱溶媒は延伸と同時に行うこともできる。また、脱溶媒は、フィルムを延伸させた後に行ってもよい。　 Drying and / or desolvation is performed by at least one means selected from, for example, vacuum drying, hot air drying, air drying, and immersion in liquid. The immersion in the liquid may be an alcohol solution such as water, a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol, or 2-propanol, or a cast film may be immersed in a mixed solution of water and alcohol to remove the solvent. good. The temperature of the desolvent solution (coagulant solution) is preferably 0 to 90 ° C. It is preferable to remove the solvent as much as possible. When the film is stretched in a liquid, desolvation can be performed at the same time as stretching. Further, the solvent removal may be performed after the film is stretched.
乾燥及び／又は脱溶媒後の未延伸フィルムは、水中で１軸延伸又は２軸延伸することができる。２軸延伸は、逐次延伸でも同時２軸延伸でもよい。２段以上の多段延伸をしてもよい。延伸倍率は、縦、横ともに、好ましくは１．０１～６倍、より好ましくは１．０５～４倍である。この範囲であると応力－歪のバランスがとりやすい。水中延伸は、２０～９０℃の水温で行われることが好ましい。延伸後のフィルムは、５０～２００℃の乾熱で５～６００秒間熱固定することが好ましい。この熱固定により、常温における寸法安定性が得られる。なお、１軸延伸したフィルムは１軸配向フィルムとなり、２軸延伸したフィルムは２軸配向フィルムとなる。　 The unstretched film after drying and / or desolving can be uniaxially stretched or biaxially stretched in water. The biaxial stretching may be sequential stretching or simultaneous biaxial stretching. Two or more stages of multi-stage stretching may be performed. The draw ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times in both the vertical and horizontal directions. Within this range, the stress-strain balance is easy to achieve. The underwater stretching is preferably performed at a water temperature of 20 to 90 ° C. The stretched film is preferably heat-fixed at 50 to 200 ° C. for 5 to 600 seconds. By this heat fixing, dimensional stability at room temperature can be obtained. The uniaxially stretched film is a uniaxially oriented film, and the biaxially stretched film is a biaxially oriented film. It was
フィルムは、カラーフィルムであってもよい。この場合、染料などの着色剤を例えばＤＭＳＯ溶媒に溶解又は分散させてＤＭＳＯ着色液を作製し、この着色液とドープ液とを混合して得られた溶液を、上述したのと同様にキャスト成形によりフィルムを作製する。その後、乾燥及び／又は脱溶媒して未延伸着色フィルムにするか、又は延伸して延伸フィルムとする。カラーフィルムは、反射板、マーカー、紫外線防止膜、スリット糸などに応用できる。　 The film may be a color film. In this case, a colorant such as a dye is dissolved or dispersed in a DMSO solvent, for example, to prepare a DMSO coloring liquid, and the solution obtained by mixing the coloring liquid and the doping liquid is cast-molded in the same manner as described above. To make a film. Then, it is dried and / or desolvated to obtain an unstretched colored film, or stretched to obtain a stretched film. The color film can be applied to a reflector, a marker, an ultraviolet protection film, a slit thread, and the like. It was
フィルムの厚さは、用途等に応じて調節することができ、例えば、1～100μｍである。　 The thickness of the film can be adjusted according to the intended use and the like, and is, for example, 1 to 100 μm. It was
本発明のポリペプチドＩは、必要に応じて他の高分子とともに溶媒に溶解又は分散して溶液又は分散液とし、溶液又は分散液を抄紙等により不織布とすることができる。或いは、ポリペプチドＩと接着剤、溶媒、他の高分子などを含む組成物を原料とし、乾式法、湿式法、スパンボンド法、メルトブローン法、サーマルボンド法、ケミカルボンド法(含浸法、スプレー法)、ニードルパンチ法、水流絡合法などにより不織布を形成することもできる。　 The polypeptide I of the present invention can be dissolved or dispersed in a solvent together with other polymers as needed to form a solution or dispersion, and the solution or dispersion can be made into a nonwoven fabric by papermaking or the like. Alternatively, a composition containing polypeptide I, an adhesive, a solvent, another polymer, etc. is used as a raw material, and a dry method, a wet method, a spunbond method, a melt blown method, a thermal bond method, or a chemical bond method (impregnation method, spray method) is used. ), Needle punching method, water flow entanglement method, etc. can also be used to form the non-woven fabric. It was
不織布の厚さは、用途等に応じて調節することができ、例えば、０．１μｍ～２ｍｍ、又は０．１μｍ～５００μｍであってもよい。好ましくは１～１００μｍである。 The thickness of the nonwoven fabric can be adjusted according to the intended use and the like, and may be, for example, 0.1 μm to 2 mm or 0.1 μm to 500 μm. It is preferably 1 to 100 μm.
次に実施例を示して本発明をより具体的に説明する。なお、本発明は実施例によって制限されるものではない。　 Next, the present invention will be described in more detail with reference to examples. The present invention is not limited to the examples. It was
参考例１（１）プラスミド発現株の作製　Trichonephila clavata（ジョロウグモ)由来のフィブロインの塩基配列及びアミノ酸配列に基づき、クモ糸タンパク質MaSp2を設計した。なお、MaSp2をコードする塩基配列は、生産性の向上を目的としてアミノ酸残基の置換、挿入及び欠失を施したアミノ酸配列(配列番号３)をコードする。なお、配列番号３において、２番目から７番目の６個のHisはHisタグであり、８番目から１２番目のSSGSSはリンカー配列である。また、１３番目から１８番目のLEVLFQはプロテアーゼ認識配列であり、１９番目から２０番目のGPはリンカー配列であり、２１番目から５４０番目がMaSp2のコーディング領域である。プロテアーゼ認識配列は、配列番号３の18番目のGlnと19番目のGlyの間で切断されるので、プロテアーゼで切断されたときに19番目から540番目の522個のアミノ酸を含むポリペプチドが生成する。　 Reference Example 1 (1) Preparation of plasmid expression strain The spider silk protein MaSp2 was designed based on the base sequence and amino acid sequence of fibroin derived from Trichonephila clavata (Trichonephila clavata). The base sequence encoding MaSp2 encodes an amino acid sequence (SEQ ID NO: 3) in which amino acid residues have been substituted, inserted or deleted for the purpose of improving productivity. In SEQ ID NO: 3, the 6th Hiss from the 2nd to the 7th are His tags, and the SSGSSs from the 8th to the 12th are linker sequences. The 13th to 18th LEVLFQ is a protease recognition sequence, the 19th to 20th GP is a linker sequence, and the 21st to 540th is a MaSp2 coding region. The protease recognition sequence is cleaved between 18th Gln and 19th Gly of SEQ ID NO: 3, so when cleaved with protease, a polypeptide containing 522 amino acids 19th to 540th is produced. .. It was
次に、MaSp2をコードする核酸を合成した。当該核酸には、５’末端にＮｄｅＩサイト及び終止コドン下流にＥｃｏＲＩサイトを付加した。当該核酸をクローニングベクター（ｐＵＣ１１８）にクローニングした。その後、同核酸をＮｄｅＩ及びＥｃｏＲＩで制限酵素処理して切り出した後、タンパク質発現ベクターｐＥＴ－２２ｂ（＋）に組換えて発現ベクターを得た。　 Next, the nucleic acid encoding MaSp2 was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector. It was
（２）タンパク質の発現 （MaSp2）　MaSp2をコードする核酸を含むｐＥＴ２２ｂ（＋）発現ベクターで、大腸菌ＢＬＲ（ＤＥ３）を形質転換した。当該形質転換大腸菌を、アンピシリンを含む２ｍＬのＬＢ培地で１５時間培養した。当該培養液を、アンピシリンを含む１００ｍＬのシード培養用培地（表１）にＯＤ が０．００５となるように添加した。培養液温度を３０℃に保ち、ＯＤ が５になるまでフラスコ培養を行い（約１５時間）、シード培養液を得た。　 (2) Protein expression (MaSp2) Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding MaSp2. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture broth was added to 100 mL of seed culture medium (Table 1) containing ampicillin so that the OD was 0.005. The culture solution temperature was kept at 30 ° C., and flask culture was carried out until the OD reached 5 (about 15 hours) to obtain a seed culture solution. It was
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
当該シード培養液を５００ｍＬの生産培地（表２）を添加したジャーファーメンターにＯＤ が０．０５となるように添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにした。　 The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 2) was added so that the OD was 0.05. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration. It was
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
生産培地中のグルコースが完全に消費された直後に、フィード液（グルコース４５５ｇ／１Ｌ、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　１２０ｇ／１Ｌ）を１ｍＬ／分の速度で添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにし、２０時間培養を行った。その後、１Ｍのイソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を培養液に対して終濃度１ｍＭになるよう添加し、MaSp2を発現誘導させた。ＩＰＴＧ添加後２０時間経過した時点で、培養液を遠心分離し、菌体を回収した。ＩＰＴＧ添加前とＩＰＴＧ添加後の培養液から調製した菌体を用いてＳＤＳ－ＰＡＧＥを行い、ＩＰＴＧ添加に依存した目的とするMaSp2サイズのバンドの出現により、目的とするMaSp2の発現を確認した。（３）タンパク質の精製（MaSp2）　ＩＰＴＧを添加してから２時間後に回収した菌体を２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｂｕｆｆｅｒ（ｐＨ７．４）で洗浄した。洗浄後の菌体を約１ｍＭのＰＭＳＦを含む２０ｍＭＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）に懸濁させ、高圧ホモジナイザー（ＧＥＡ　Ｎｉｒｏ　Ｓｏａｖｉ社製）で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで２０ｍＭＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）で洗浄した。洗浄後の沈殿物を１００ｍｇ／ｍＬの濃度になるように８Ｍ　グアニジン緩衝液（８Ｍグアニジン塩酸塩、１０ｍＭリン酸二水素ナトリウム、２０ｍＭＮａＣｌ、１ｍＭＴｒｉｓ－ＨＣｌ、ｐＨ７．０）で懸濁し、６０℃で３０分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ（三光純薬株式会社製のセルロースチューブ３６／３２）を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収し、凍結乾燥機で水分を除き、凍結乾燥粉末を回収することにより、クモ糸タンパク質MaSp2を得た。　 Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of MaSp2. Twenty hours after the addition of IPTG, the culture broth was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture broth before and after the addition of IPTG, and the expression of the target MaSp2 was confirmed by the appearance of the target MaSp2 size band dependent on the addition of IPTG. (3) Protein purification (MaSp2) The cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 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 crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 30 at 60 ° C. Stir for 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 dialysis was recovered by centrifugation, water was removed by a freeze-dryer, and the freeze-dried powder was recovered to obtain spider silk protein MaSp2. It was
実施例１（１）タンパク質の合成 （ポリペプチドＩ）　Tｒｉｃｈｏｎｅｐｈｉｌａ　ｃｌａｖａｔａ（ジョロウグモ)の前身及び絹糸腺を使用したトランスクリプトーム解析により、絹糸腺末端部で比較的高発現の転写物を認めた。この転写物がコードする機能未知タンパク質の塩基配列及びアミノ酸配列に基づき、配列番号１で示されるアミノ酸配列を有するクモ糸タンパク質を設計した。なお、配列番号１で示されるアミノ酸配列は、ジョロウグモ由来の機能未知タンパク質のアミノ酸配列に対して、シグナル配列を除去し、Ｎ末端にHisタグ配列(HHHHHH)、リンカー配列（SSGSS）、プロテアーゼ認識サイト（LEVLFQGP）が付加されている。　 Example 1 (1) Protein synthesis (Polypeptide I) Transcriptome analysis using the predecessor of Trichonephila clavata (Joro spider) and the silk gland revealed a relatively highly expressed transcript at the end of the silk gland. Based on the base sequence and amino acid sequence of the protein of unknown function encoded by this transcript, a spider silk protein having the amino acid sequence shown by SEQ ID NO: 1 was designed. The amino acid sequence shown by SEQ ID NO: 1 has a signal sequence removed from the amino acid sequence of a protein of unknown function derived from Joro spider, and has a His tag sequence (HHHHHH), a linker sequence (SSGSS), and a protease recognition site at the N-terminal. (LEVLFQGP) is added. It was
次に、ポリペプチドＩをコードする核酸配列(配列番号２)を合成した。当該核酸には、５’末端にＮｄｅＩサイト及び終止コドン下流にＥｃｏＲＩサイトを付加した。当該核酸をクローニングベクター（ｐＵＣ１１８）にクローニングした。その後、同核酸をＮｄｅＩ及びＥｃｏＲＩで制限酵素処理して切り出した後、タンパク質発現ベクターｐＥＴ－２２ｂ（＋）に組換えて発現ベクターを得た。　 Next, a nucleic acid sequence encoding polypeptide I (SEQ ID NO: 2) was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector. It was
（２）タンパク質の発現 （ポリペプチドＩ）　配列番号１で示されるアミノ酸配列を有するタンパク質をコードする核酸を含むｐＥＴ２２ｂ（＋）発現ベクターで、大腸菌ＢＬＲ（ＤＥ３）を形質転換した。当該形質転換大腸菌を、アンピシリンを含む２ｍＬのＬＢ培地で１５時間培養した。当該培養液を、アンピシリンを含む１００ｍＬのシード培養用培地（表３）にＯＤ が０．００５となるように添加した。培養液温度を３０℃に保ち、ＯＤが５になるまでフラスコ培養を行い（約１５時間）、シード培養液を得た。　 (2) Protein expression (Polypeptide I) Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence shown in SEQ ID NO: 1. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture broth was added to 100 mL of seed culture medium (Table 3) containing ampicillin so that the OD was 0.005. The culture solution temperature was maintained at 30 ° C., and flask culture was carried out until the OD reached 5 (about 15 hours) to obtain a seed culture solution. It was
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
当該シード培養液を５００ｍＬの生産培地（表４）を添加したジャーファーメンターにＯＤが０．０５となるように添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにした。　 The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 4) was added so that the OD was 0.05. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration. It was
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
生産培地中のグルコースが完全に消費された直後に、フィード液（グルコース４５５ｇ／１Ｌ、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　１２０ｇ／１Ｌ）を１ｍＬ／分の速度で添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにし、２０時間培養を行った。その後、１Ｍのイソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を培養液に対して終濃度１ｍＭになるよう添加し、ポリペプチドＩを発現誘導させた。ＩＰＴＧ添加後２０時間経過した時点で、培養液を遠心分離し、菌体を回収した。ＩＰＴＧ添加前とＩＰＴＧ添加後の培養液から調製した菌体を用いてＳＤＳ－ＰＡＧＥを行い、ＩＰＴＧ添加に依存した目的とする配列番号１で示されるポリペプチドＩのバンドの出現により、目的タンパク質の発現を確認した。　 Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of polypeptide I. Twenty hours after the addition of IPTG, the culture broth was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture broth before and after the addition of IPTG, and the appearance of the band of the polypeptide I represented by the target SEQ ID NO: 1 depending on the addition of IPTG resulted in the addition of the target protein. Expression was confirmed. It was
（２）タンパク質の精製（ポリペプチドＩ）　ＩＰＴＧを添加してから２時間後に回収した菌体を1mMのPMSF, 1mMのDTT、7.5Mの尿素を含むリン酸ナトリウム緩衝液（50mM リン酸ナトリウム、300mM塩化ナトリウム、7.5M尿素、ｐＨ７）に懸濁し、高圧ホモジナイザー（ＧＥＡ　Ｎｉｒｏ　Ｓｏａｖｉ社製）で細胞を破砕した。破砕した細胞を遠心分離した後に0.44 μm のフィルターでろ過し、上清を得た。この上清をNi-NTA (バイオラッド社製)に負荷し、15mM イミダゾール、1mMのDTT、7.5Mの尿素を含むリン酸ナトリウム緩衝液（ｐH７）でNi-NTAを十分洗浄した後、500mM イミダゾール、1mMのDTT、7.5Mの尿素を含むリン酸ナトリウム緩衝液（ｐH７）を用いてポリペプチドＩを溶出させて回収した。得られた ポリペプチドＩ が含まれる画分を7.5Mの尿素、1mMのDTT、50mMの塩化ナトリウムを含む２０ｍＭＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）に対して透析を行い、緩衝液を交換した。この溶液を陰イオン交換カラム（バイオラッド社製）に対して負荷し、7.5Mの尿素、1mMのDTT、80mMの塩化ナトリウムを含む２０ｍＭＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）で十分に洗浄した後、7.5Mの尿素、1mMのDTT、1Mの塩化ナトリウムを含む２０ｍＭ Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）を用いてポリペプチドＩを溶出させて回収した。得られた ポリペプチドＩ が含まれる画分を透析チューブを用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収し、凍結乾燥機で水分を除き、凍結乾燥粉末を回収することにより、ポリペプチドＩの粉末を得た。ポリペプチドＩのＳＤＳＰＡＧＥの結果、分子量は約２６．３ｋＤａであることが明らかになった。　 (2) Protein purification (Polypeptide I) 2 hours after the addition of IPTG, the collected cells were collected with 1 mM PMSF, 1 mM DTT, and 7.5 M sodium phosphate buffer (50 mM sodium phosphate, The cells were suspended in 300 mM sodium chloride, 7.5 M urea, pH 7), and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). After centrifuging the crushed cells, the cells were filtered through a 0.44 μm filter to obtain a supernatant. This supernatant is loaded onto Ni-NTA (manufactured by Biorad), and Ni-NTA is thoroughly washed with sodium phosphate buffer (pH 7) containing 15 mM imidazole, 1 mM DTT, and 7.5 M urea, and then 500 mM imidazole. Polypeptide I was eluted and recovered using sodium phosphate buffer (pH 7) containing 1 mM DTT and 7.5 M urea. The fraction containing the obtained polypeptide I was dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 7.5 M urea, 1 mM DTT, and 50 mM sodium chloride, and the buffer was replaced. This solution was loaded onto an anion exchange column (Biorad) and washed thoroughly with 20 mM Tris-HCl buffer (pH 7.4) containing 7.5 M urea, 1 mM DTT, and 80 mM sodium chloride. Polypeptide I was recovered by eluting with 20 mM Tris-HCl buffer (pH 7.4) containing 7.5 M urea, 1 mM DTT, and 1 M sodium chloride. The fraction containing the obtained polypeptide I was dialyzed against water using a dialysis tube. The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed by a lyophilizer, and the lyophilized powder was recovered to obtain a powder of Polypeptide I. As a result of SDSPAGE of polypeptide I, it was revealed that the molecular weight was about 26.3 kDa. It was
遠沈管に添加したヘキサフルオロイソプロパノール（ＨＦＩＰ） 8ml にクモ糸タンパク質MaSp2 を表５に従い添加、撹拌し、ポリペプチドＩ（配列番号１）を表５に従い添加し、温調器を用い60℃、2時間で加熱撹拌し、溶液中の全
タンパク質質量に対し、ポリペプチドＩが質量0%、1％、3％、５％となるよう調製したタンパク質溶液を得た。　 Add spider protein MaSp2 to 8 ml of hexafluoroisopropanol (HFIP) added to the centrifuge tube according to Table 5, stir, add polypeptide I (SEQ ID NO: 1) according to Table 5, and use a temperature controller at 60 ° C., 2 The mixture was heated and stirred for a period of time to obtain a protein solution prepared so that the weight of polypeptide I was 0%, 1%, 3%, and 5% with respect to the total protein mass in the solution.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
加熱してクモ糸タンパク質を溶解させた後、遠沈管を開封し、シャーレ上にタンパク質溶液を展開した状態で、室温で１６時間静置して乾燥し、クモ糸タンパク質フィルムを得た。　 After heating to dissolve the spider silk protein, the centrifuge tube was opened, and the protein solution was developed on a petri dish and allowed to stand at room temperature for 16 hours to dry to obtain a spider silk protein film. It was
クモ糸タンパク質フィルムの物性測定　クモ糸タンパク質フィルムを、長方形に切り出し、試験片（５ｍｍ×１５ｍｍ）とした。試験片を用い、島津製作所社製引張試験装置EZ-LX HSにより、引張試験を実施した。引張試験により、破断点変位（ひずみ）（％）、最大点応力（ＭＰａ）、ヤング率（ＭＰａ）、タフネス（［ＭＪ］／［ｍ３］）を測定した。各サンプルについて５回行った測定の平均値を表６に示す。タフネスは、下記式により求められる。応力［Ｎ／ｍ２］×歪み［ｍｍ／ｍｍ］＝エネルギー［Ｎｍ］／体積［ｍ３］＝エネルギー［Ｊ］／体積［ｍ３］　 Measurement of Physical Properties of Spider Thread Protein Film The spider silk protein film was cut into a rectangular shape and used as a test piece (5 mm × 15 mm). A tensile test was carried out using a test piece using a tensile test device EZ-LX HS manufactured by Shimadzu Corporation. The fracture point displacement (strain) (%), maximum point stress (MPa), Young's modulus (MPa), and toughness ([MJ] / [m 3 ]) were measured by a tensile test. Table 6 shows the average value of the measurements taken 5 times for each sample. Toughness is calculated by the following formula. Stress [N / m 2 ] x strain [mm / mm] = energy [Nm] / volume [m 3 ] = energy [J] / volume [m 3 ]
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
破断点変位が高いほど、伸度に優れる（高い伸度を有する）ことを示す。最大点応力（ＭＰａ）が高いほど、応力に優れる（高い応力を有する）ことを示す。タフネス（［ＧＪ］／［ｍ３］）が高いほど、靭性に優れることを示し、ポリペプチドＩが添加されていないフィルムとポリペプチドＩが添加されたクモ糸タンパク質フィルムを比較するとポリペプチドＩが添加されたクモ糸タンパク質フィルムの方が優れた最大点応力（ＭＰａ）、タフネス（［ＧＪ］／［ｍ３］）を有していることが確認された。　 The higher the displacement at the breaking point, the better the elongation (has a higher elongation). The higher the maximum point stress (MPa), the better the stress (has a higher stress). The higher the toughness ([GJ] / [m 3 ]), the better the toughness, and when comparing the film without polypeptide I and the spider silk protein film with polypeptide I, polypeptide I is found. It was confirmed that the added spider silk protein film had superior maximum point stress (MPa) and toughness ([GJ] / [m 3]).
クモ糸タンパク質フィルムの透過度の測定　上記で得られたクモ糸タンパク質フィルムを、分光光度計（JASCO社製 紫外可視分光光度計 V-750ST）を用い、フィルムの透過性を測定した。測定結果は表７に示す。　 Measurement of permeability of spider silk protein film The transmittance of the spider silk protein film obtained above was measured using a spectrophotometer (JASCO's ultraviolet-visible spectrophotometer V-750ST). The measurement results are shown in Table 7. It was
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
測定値 T% (500nm) の数値が高いほど透過性に優れたことを示し、ポリペプチドＩが添加されていないフィルムとポリペプチドＩが添加されたクモ糸タンパク質フィルムを比較するとポリペプチドＩが添加されたクモ糸タンパク質フィルムの方が優れた透過性を有していることが確認された。　 The higher the measured value T% (500 nm), the better the permeability, and when comparing the film without polypeptide I and the spider silk protein film with polypeptide I added, polypeptide I was added. It was confirmed that the spider thread protein film obtained had better permeability. It was
クモ糸タンパク質繊維の製造　LiClが4質量パーセントになるよう調製したＤＭＳＯ5.89gに、クモ糸タンパク質MaSp2を1.66ｇ添加し、温調器を用い90℃、8時間で加熱攪拌し、タンパク質濃度22質量パーセントのタンパク質溶液を得た。　 Production of spider silk protein fiber 1.66 g of spider silk protein MaSp2 was added to DMSO 5.89 g prepared so that LiCl was 4% by mass, and heated and stirred at 90 ° C for 8 hours using a temperature controller, and the protein concentration was 22 mass. A percentage protein solution was obtained. It was
LiClが4質量パーセントになるよう調製したＤＭＳＯ4.2gに、クモ糸タンパク質MaSp2を1.188g及びポリペプチドＩを0.012g添加し、温調器を用い90℃、8時間で加熱攪拌し、タンパク質濃度22質量パーセントのタンパク質溶液を得た。得られたタンパク質溶液中のゴミと泡を取り除き、ドープ液とした(表８)。　 To 4.2 g of DMSO prepared to have LiCl of 4% by mass, 1.188 g of spider silk protein MaSp2 and 0.012 g of polypeptide I were added, and the mixture was heated and stirred at 90 ° C. for 8 hours using a temperature controller, and the protein concentration was 22. A mass percent protein solution was obtained. Dust and bubbles in the obtained protein solution were removed to prepare a dope solution (Table 8). It was
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
上記のようにして得られたドープ液と図１に示される自社製の紡糸装置を用いて公知の乾湿式紡糸を行って、クモ糸タンパク質繊維をボビンに巻きとった。なお、ここでは、乾湿式紡糸を下記の条件で行った。凝固液（メタノール）の温度：５～１０℃　延伸倍率：５．５倍、６．０倍乾燥温度：70℃　 A known dry-wet spinning was performed using the doping solution obtained as described above and the in-house spinning apparatus shown in FIG. 1, and spider silk protein fibers were wound around a bobbin. Here, dry-wet spinning was performed under the following conditions. Coagulant (methanol) temperature: 5 to 10 ° C Stretching ratio: 5.5 times, 6.0 times Drying temperature: 70 ° C
クモ糸タンパク質の物性測定　６０ｃｍの長さに切り出したクモ糸タンパク質繊維サンプルを4つ（ｎ＝20）用意し、インストロン社製引張試験機（Ｍ１０－１６２７９－ＥＮ）を用い、その最大引張応力を測定した。試験条件は、以下のとおりである。引張速度：１０ｃｍ／分の速度ロードセル：１０Ｎ相対湿度：６５％温度：２０℃測定時のつかみ治具間距離：１５ｃｍつかみ冶具：クリップ式　 Measurement of physical properties of spider silk protein Prepare four spider silk protein fiber samples (n = 20) cut out to a length of 60 cm, and use an Instron tensile tester (M10-16279-EN) to obtain the maximum tensile stress. Was measured. The test conditions are as follows. Tensile speed: 10 cm / min Speed load cell: 10N Relative humidity: 65% Temperature: 20 ° C Distance between grip jigs when measuring: 15 cm Grasp jig: Clip type
試験結果は表９に示す。表９は、引張試験結果から求めたそれぞれのサンプルの引張強度、破断点変位、ヤング率、繊維径、遠心倍率の値を示す。　 The test results are shown in Table 9. Table 9 shows the values of tensile strength, fracture point displacement, Young's modulus, fiber diameter, and centrifugal magnification of each sample obtained from the tensile test results. It was
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
ポリペプチドＩが添加されていないクモ糸タンパク質繊維とポリペプチドＩが添加されたクモ糸タンパク質繊維を比較するとポリペプチドＩが添加されたクモ糸タンパク質繊維の方の伸度が向上していることが確認された。 Comparing the spider silk protein fiber to which polypeptide I is not added and the spider silk protein fiber to which polypeptide I is added, it can be seen that the elongation of the spider silk protein fiber to which polypeptide I is added is improved. confirmed.
１　押出し装置２　未延伸糸製造装置３　湿熱延伸装置４　乾燥装置６　ドープ液１０　紡糸装置２０　凝固液槽２１　延伸浴槽３６　タンパク質フィラメント 1 Extruding device 2 Undrawn yarn manufacturing device 3 Wet and heat stretching device 4 Drying device 6 Doping liquid 10 Spinning device 20 Coagulation liquid tank 21 Stretching bath 36 Protein filament

Claims (12)

  1. 配列番号１の21番目から252番目のアミノ酸配列と７０％以上の同一性を有するポリペプチド。 A polypeptide having 70% or more identity with the 21st to 252nd amino acid sequences of SEQ ID NO: 1.
  2. 請求項１のポリペプチドをコードする核酸またはその相補鎖。 A nucleic acid encoding the polypeptide of claim 1 or a complementary strand thereof.
  3. 請求項１に記載のポリペプチドを含む成形体 A molded product containing the polypeptide according to claim 1.
  4. 前記成形体が繊維又はフィルムである、請求項３に記載の成形体。 The molded product according to claim 3, wherein the molded product is a fiber or a film.
  5. 請求項１に記載のポリペプチドと、溶媒及び他の高分子からなる群から選ばれる少なくとも１種を含む、組成物。 A composition comprising the polypeptide according to claim 1 and at least one selected from the group consisting of a solvent and other macromolecules.
  6. 前記ポリペプチドの含有量が前記組成物の質量に対して1％以上である、請求項５に記載の組成物。 The composition according to claim 5, wherein the content of the polypeptide is 1% or more with respect to the mass of the composition.
  7. 前記組成物がHFIP(ヘキサフルオロイソプロパノール)、DMSO（ジメチルスルホキシド）及びギ酸からなる群から選ばれる少なくとも１種の溶媒を含む、請求項５又は６に記載の組成物。 The composition according to claim 5 or 6, wherein the composition comprises at least one solvent selected from the group consisting of HFIP (hexafluoroisopropanol), DMSO (dimethyl sulfoxide) and formic acid.
  8. 前記他の高分子が構造タンパク質である請求項５～７のいずれか１項に記載の組成物。 The composition according to any one of claims 5 to 7, wherein the other polymer is a structural protein.
  9. 前記構造タンパク質がフィブロインである、請求項８に記載の組成物。 The composition according to claim 8, wherein the structural protein is fibroin.
  10. 前記フィブロインがクモ糸タンパク質である、請求項９に記載の組成物。 The composition according to claim 9, wherein the fibroin is a spider silk protein.
  11. 請求項１のポリペプチドを含む、成形体の物性向上剤。 An agent for improving the physical properties of a molded product, which comprises the polypeptide of claim 1.
  12. 配列番号１の21番目から252番目のアミノ酸配列と７０％以上の同一性を有するポリペプチド含む組成物を製造する方法であって、前記ポリペプチドと他の高分子とを混合する工程を含む組成物の製造方法。
          A method for producing a composition containing a polypeptide having 70% or more identity with the 21st to 252nd amino acid sequences of SEQ ID NO: 1, which comprises a step of mixing the polypeptide with another polymer. Manufacturing method of things.
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHAW, R.C. ET AL.: "Proteomic Evidence for Components of Spider Silk Synthesis from Black Widow Silk Glands and Fibers", J. PROTEOME RES., vol. 14, no. 10, 2015, pages 4223 - 4231, XP055875177 *
CLARKE THOMAS H., GARB JESSICA E, HAYASHI CHERYL Y, HANEY ROBERT A, LANCASTER ALEXANDER K, CORBETT SUSAN, AYOUB NADIA A: "Multi-tissue transcriptomics of the black widow spider reveals expansions, co-options, and functional processes of the silk gland gene toolkit", BMC GENOMICS, vol. 15, no. 365, 2014, pages 1 - 17, XP021187307 *
GARB JESSICA E., HANEY ROBERT A., SCHWAGER EVELYN E., GREGORIČ MATJAŽ, KUNTNER MATJAŽ, AGNARSSON INGI, BLACKLEDGE TODD A.: "The transcriptome of Darwin' s bark spider silk glands predicts proteins contributing to dragline silk toughness", COMMUNICATIONS BIOLOGY, vol. 2, no. 275, 2019, pages 1 - 8, XP055875220 *
KONO, NOBUAKI ET AL.: "Orb-weaving spider Araneus ventricosus genome elucidates the spidroin gene catalogue", SCIENTIFIC REPORTS, vol. 9, no. 8380, 2019, pages 1 - 13, XP055875203 *
WHAITE, A.D. ET AL.: "Major ampullate silk gland transcriptomes and fibre proteomes of the golden orbweavers, Nephila plumipes and Nephila pilipes (Araneae: Nephilidae", PLOS ONE, vol. 13, no. 10, 2018, pages 1 - 22, XP055875154 *

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