WO2020067514A1 - Fibre de fibroïne de soie d'araignée modifiée et son procédé de production - Google Patents

Fibre de fibroïne de soie d'araignée modifiée et son procédé de production Download PDF

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WO2020067514A1
WO2020067514A1 PCT/JP2019/038375 JP2019038375W WO2020067514A1 WO 2020067514 A1 WO2020067514 A1 WO 2020067514A1 JP 2019038375 W JP2019038375 W JP 2019038375W WO 2020067514 A1 WO2020067514 A1 WO 2020067514A1
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silk fibroin
spider silk
amino acid
seq
modified spider
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Japanese (ja)
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オリバ－ セイエッド シャファ－ト
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Spiber Inc
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Spiber Inc
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/02—Elements
    • C08K3/04—Carbon
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K7/00—Use of ingredients characterised by shape
    • 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
    • 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 modified spider silk fibroin fiber and a method for producing the same.
  • the present invention also relates to a dispersion that can be used for producing the modified spider silk fibroin fiber.
  • the present invention also relates to products comprising the modified fibroin fibers described above.
  • fibroin fiber is attracting attention as a new material with high utility value in the future.
  • Conventionally, regenerated silk fibroin fibers and spider silk fibroin fibers are known as fibroin fibers.
  • Many methods for producing small-diameter fibroin fibers have been reported.
  • Non-Patent Document 2 a method of obtaining fibers having a diameter of 2 ⁇ m by forming fibers from the above-mentioned dope solution using a microfluidic device, and then drawing and impregnating the fibers in an aqueous alcohol solution. Have been.
  • Patent Document 1 a method has been reported in which a fiber having an average diameter of 1 ⁇ m or less is obtained by discharging a spider silk fibroin dope solution from a spinneret to which a voltage is applied by electrospinning.
  • an object of the present invention is to provide a small-diameter fibroin fiber by a simple method.
  • the present invention relates to, for example, the following inventions.
  • the polar solvent is hexafluoroisopropanol, hexafluoroacetone, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile
  • the dispersion according to [1] which is at least one selected from the group consisting of N-methylmorpholine N-oxide, formic acid, ethylene glycol, tetrahydrofuran, and water.
  • planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide.
  • liquid [4] A modified spider silk fibroin, a carbon material, and a polar solvent, The carbon material is carbon black nanoparticles, The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran.
  • a carbon material dispersion which is at least one selected from the group consisting of: [5] The dispersion according to any one of [1] to [4], wherein the content of the carbon material is 25 parts by mass or less based on 100 parts by mass of the modified spider silk fibroin. [6] The dispersion according to any one of [1] to [5], which is a dope. [7] A modified spider yarn comprising a modified spider yarn fibroin and at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles, and having an average fiber diameter of 3 ⁇ m or less. Fibroin fiber.
  • Spider silk fibroin fiber [10] A product comprising the modified spider silk fibroin fiber according to any one of [7] to [9].
  • Modified spider silk fibroin a carbon material, comprising a step of forming fibrils by a dry spinning method from a dispersion containing a polar solvent, wherein the carbon material is carbon black nanoparticles, production of modified spider silk fibroin fibers Method.
  • [16] Modified spider silk fibroin, a carbon material, and a polar solvent, a step of forming fibrils from a dispersion liquid, Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils, A method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is one or more layers of planar graphene.
  • Modified spider silk fibroin a carbon material, a polar solvent, a step of forming fibrils by a dry spinning method from a dispersion containing, Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils, And a method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is carbon black nanoparticles.
  • Modified spider silk fibroin comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent, A method for dispersing a carbon material, wherein the carbon material is one or two or more layers of planar graphene.
  • Modified spider silk fibroin comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent
  • the carbon material is carbon black nanoparticles
  • the polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran.
  • a method for dispersing a carbon material which is at least one selected from the group.
  • a dispersion aid for dispersing a carbon material in a polar solvent comprising a modified spider silk fibroin, wherein the carbon material is one or more layers of planar graphene.
  • a modified auxiliary agent for dispersing a carbon material in a polar solvent comprising a modified spider silk fibroin, The carbon material is carbon black nanoparticles,
  • the polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. At least one selected from dispersing aids.
  • a thinning agent for producing a modified spider silk fibroin fiber having a reduced diameter comprising at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles.
  • the carbon material dispersion liquid of the present embodiment includes a modified spider silk fibroin, a carbon material, and a polar solvent, and the carbon material is one or two or more layers of planar graphene.
  • the term “dispersion” refers to a precipitate, an aggregate, or a precipitate and an aggregate that is visually free from a precipitate, an aggregate, or a precipitate and an aggregate even after standing still for at least 7 days. Refers to a solution without any objects.
  • the carbon material dispersion of the present invention is excellent in dispersibility of the carbon material, can sufficiently suppress aggregation and precipitation of the carbon material, and is excellent in operability.
  • the dispersion of this embodiment can be used as it is, or appropriately concentrated or diluted, as a spinning stock solution (dope solution) for producing a modified spider silk fibroin fiber.
  • the carbon material dispersion liquid of the present embodiment has good dispersibility and excellent operability, it can be suitably used for uses other than the raw material of the modified spider silk fibroin fiber of the present invention. For example, it can be suitably used for producing a composite material containing a carbon material.
  • planar graphene refers to graphene and graphene analogs that form a two-dimensional sheet-like molecular structure with contained carbon atoms. Therefore, carbon nanotubes that form a cylindrical structure with carbon atoms and fullerenes that form a spherical structure are excluded from “planar graphene”.
  • the “planar graphene” may have a two-dimensional sheet-like structure in a part of its molecular structure, and the entire molecular structure may not be planar.
  • the planar graphene may have a two-dimensional sheet-like molecular structure of one layer or two or more layers.
  • planar graphene examples include graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, reduced functionalized graphene oxide, and the like. Among these, graphene oxide is preferable.
  • the degree of oxidation of graphene oxide is not particularly limited, and known graphene oxide can be used.
  • the degree of oxidation of graphene oxide may be, for example, from 4 to 55%, from 4 to 50%, from 4 to 45%, from 4 to 40%, from 4 to 35%. May be 4 to 30%, may be 4 to 25%, may be 4 to 20%, may be 4 to 15%, may be 4 to 10%.
  • the functionalized graphene oxide has a structure in which a part of the oxygen-containing functional group of the graphene oxide is chemically modified with another functional group or the like.
  • Known functionalized graphene oxide can be used.
  • the functionalized graphene oxide include alkylamine-functionalized graphene oxide, ammonia-functionalized graphene oxide, amine-functionalized graphene oxide, thiol-functionalized graphene oxide, alkyne-functionalized graphene oxide, and glucose-functionalized graphene oxide.
  • the carbon / oxygen ratio of the reduced graphene oxide is not particularly limited, and a known reduced graphene oxide can be used.
  • the carbon / oxygen ratio of the reduced graphene oxide may be, for example, 90/10 or 75/35.
  • the functionalized reduced graphene oxide has a structure in which a part of the oxygen-containing functional group of reduced graphene oxide is chemically modified with another functional group or the like.
  • Known functionalized reduced graphene oxide can be used.
  • Examples of the functionalized reduced graphene oxide include amine-functionalized reduced graphene oxide, octadecylamine-functionalized reduced graphene oxide, piperazine-functionalized reduced graphene oxide, tetraethylenepentamine-functionalized reduced graphene oxide, and glucose-modified reduction.
  • Graphene oxide and the like are examples of the functionalized reduced graphene oxide.
  • planar graphene in the form of powder, sheet, liquid or the like can be used.
  • planar graphene in a powder or liquid form it is preferable to use planar graphene in a powder or liquid form.
  • graphene it is preferable to use planar graphene in a liquid form.
  • planar graphene in a liquid form a graphene ink in which planar graphene is dissolved or dispersed in a solvent or a dispersion medium can be used.
  • any polar solvent that can disperse or dissolve the modified spider silk fibroin can be used.
  • the polar solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), and 1,3.
  • Organic solvents such as -dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine N-oxide (NMO), formic acid, ethylene glycol and tetrahydrofuran (THF); Water and the like.
  • hexafluoroisopropanol, dimethyl sulfoxide and formic acid are more preferred, and dimethyl sulfoxide and formic acid are even more preferred.
  • These organic solvents may include water. These solvents may be used alone or as a mixture of two or more.
  • Carbon black nanoparticles In the carbon material dispersion liquid of the present embodiment, as the carbon material, carbon black nanoparticles can be used instead of or together with the planar graphene. Carbon black nanoparticles are fine particles formed of carbon. In the present embodiment, as the carbon black nanoparticles, it is preferable to use fine particles having an average particle diameter of less than 3 ⁇ m, more preferably to use fine particles of less than 2 ⁇ m, and still more preferably to use fine particles of less than 1 ⁇ m.
  • the method for producing carbon black nanoparticles is not particularly limited, and for example, using carbon black nanoparticles produced by a known production method such as a furnace method, a channel method, an acetylene method, an oil smoke method, and a pine smoke method. Can be.
  • a known production method such as a furnace method, a channel method, an acetylene method, an oil smoke method, and a pine smoke method.
  • carbon black nanoparticles are used as the carbon material
  • N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, and the like are used as polar solvents for the carbon material dispersion. Examples thereof include N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. These solvents may be used alone or as a mixture of two or more.
  • the carbon material dispersion of the present embodiment is prepared by using a modified spider silk fibroin, a carbon material, and a polar solvent.
  • the dispersion is, for example, a mixing and dispersing machine (for example, a ball mill, a bead mill, a sand mill, a roll mill, a homogenizer, an ultrasonic homogenizer, a high-pressure homogenizer, an ultrasonic device, an attritor, a dissolver, a paint shaker, etc.) in which a carbon material and a polar solvent are mixed. And then adding the modified spider silk fibroin.
  • a mixing and dispersing machine for example, a ball mill, a bead mill, a sand mill, a roll mill, a homogenizer, an ultrasonic homogenizer, a high-pressure homogenizer, an ultrasonic device, an attritor, a dissolver, a paint shaker, etc.
  • the dispersion may be prepared by adding a carbon material to a solution obtained by mixing a modified spider silk fibroin and a polar solvent using a mixing disperser, and mixing the carbon material and the polar solvent using a mixing disperser. And a solution in which the modified spider silk fibroin is dissolved in a different polar solvent.
  • the carbon material dispersion liquid of the present embodiment may be in a liquid form or a semi-solid form such as a paste or a gel, but is preferably in a liquid form.
  • the upper limit of the content of the carbon material in the carbon material dispersion of the present embodiment may be 25 parts by mass or less, 20 parts by mass or less, and 100 parts by mass of the modified spider silk fibroin. Not more than 10 parts by mass, not more than 5 parts by mass, not more than 4 parts by mass, not more than 3 parts by mass, not more than 1 part by mass, Good.
  • the lower limit of the content of the carbon material in the carbon material dispersion liquid of the present embodiment may be not less than 0.01 part by mass, and not less than 0.03 part by mass, based on 100 parts by mass of the modified spider silk fibroin.
  • it may be 0.05 parts by mass or more, may be 0.06 parts by mass or more, may be 0.07 parts by mass or more, may be 0.08 parts by mass or more, and may be 0.09 parts by mass. Parts or more.
  • the content of the carbon material in the carbon material dispersion liquid of the present embodiment may be 0.01 to 25 parts by mass, 0.05 parts by mass or more 25 parts by mass or less, may be 0.01 parts by mass or more and 20 parts by mass or less, may be 0.05 parts by mass or more and 20 parts by mass or less, 0.01 parts by mass or more and 15 parts by mass or less May be 0.05 to 15 parts by mass, 0.01 to 10 parts by mass, 0.05 to 10 parts by mass, 0.01 to 5 parts by mass, 0.05 to 5 parts by mass, 0.01 to 4 parts by mass, 0.05 or more parts by mass 4 parts by mass or less, and 0.01 parts by mass or less.
  • the amount may be 0.01 to 1 part by mass, and may be 0.05 to 1 part by mass.
  • the upper limit of the content of the carbon material in the dope solution is 1 part by mass or less based on 100 parts by mass of the modified spider silk fibroin. Is preferably 0.95 parts by mass or less, 0.9 parts by mass or less, 0.8 parts by mass or less, 0.7 parts by mass or less, and 0.1 part by mass or less. 6 parts by mass or less, 0.5 parts by mass or less, 0.4 parts by mass or less, 0.3 parts by mass or less, and 0.2 parts by mass or less. May be.
  • the lower limit of the content of the carbon material in the dope solution may be 0.01 parts by mass or more, 0.03 parts by mass or more with respect to 100 parts by mass of the modified spider silk fibroin. It may be at least 05 parts by mass, at least 0.06 parts by mass, at least 0.07 parts by mass, at least 0.08 parts by mass, at least 0.09 parts by mass. May be.
  • the content of the carbon material in the dope solution is preferably, for example, 0.01 part by mass or more and 1 part by mass or less, and preferably 0.01 part by mass or more and 1 part by mass with respect to 100 parts by mass of the modified spider silk fibroin.
  • the amount is more preferably less than 0.03 parts by mass and less than 1 part by mass, particularly preferably from 0.05 parts by mass to less than 1 part by mass, and more preferably from 0.05 parts by mass to 0.1 part by mass.
  • 95 parts by mass or less may be 0.05 parts by mass or more and 0.9 parts by mass or less, may be 0.05 parts by mass or more and 0.8 parts by mass or less, and may be 0.05 parts by mass or more and 0 parts by mass or less.
  • the concentration of the modified spider silk fibroin in the carbon material dispersion of the present embodiment may be 1 to 40% by mass, 1 to 35% by mass, and 1 to 40% by mass when the total amount of the dispersion is 100% by mass. 30% by mass, 1 to 25% by mass, 1 to 20% by mass, 1 to 15% by mass, 1 to 10% by mass, 1 to 1% by mass It may be 5% by mass, 1-3% by mass, 1-2% by mass.
  • concentration of the modified spider silk fibroin is 1% by mass or more, the dispersibility of the carbon material can be sufficiently improved.
  • the concentration of the modified spider silk fibroin is 40% by mass or less, it is possible to avoid a decrease in the dispersibility of the carbon material due to a significant increase in viscosity.
  • the concentration of the modified spider silk fibroin may be 10 to 40% by mass when the total amount of the dope solution is 100% by mass. More preferably, it is 10 to 35% by mass, more preferably 12 to 35% by mass, more preferably 15 to 35% by mass, more preferably 15 to 30% by mass, The content is more preferably 20 to 35% by mass, and particularly preferably 20 to 30% by mass.
  • the concentration of the modified spider silk fibroin is 10% by mass or more, the dope solution can be more stably discharged from the spinneret, and the productivity is improved.
  • the concentration of the modified spider silk fibroin is 40% by mass or less, it is possible to prevent the holes of the spinneret from being closed when the dope is discharged from the spinneret, thereby improving the productivity.
  • the dispersion may be stirred or shaken for a certain period of time to promote dissolution. At that time, if necessary, the dispersion may be heated to a temperature at which it can be dissolved depending on the modified spider silk fibroin and the polar solvent used. The dispersion may be heated to, for example, 30C or higher, 40C or higher, 50C or higher, 60C or higher, 70C or higher, 80C or higher, or 90C or higher. The upper limit of the heating temperature is, for example, equal to or lower than the boiling point of the polar solvent.
  • the viscosity of the dispersion of this embodiment may be set as appropriate.
  • the viscosity is not particularly limited as long as it allows dry spinning. From the viewpoint of productivity, it may be 3000 to 50,000 mPa ⁇ sec at 25 ° C. 5000 to 50000 mPa ⁇ sec, 5000 to 40000 mPa ⁇ sec, 5000 to 30000 mPa ⁇ sec, 5000 to 20000 mPa ⁇ sec, and 5000 to 15000 mPa ⁇ sec. 5000 to 12000 mPa ⁇ sec.
  • the viscosity of the spinning solution can be measured using, for example, a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
  • the dispersion of the present embodiment may further contain an inorganic salt.
  • the inorganic salt can be used as a promoter for dissolving the modified spider silk fibroin in a polar solvent.
  • the inorganic salt may be an inorganic salt composed of the following Lewis acid and Lewis base.
  • the Lewis base include a halide ion and the like.
  • Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions.
  • Examples of the inorganic salt include an alkali metal halide and an alkaline earth metal halide.
  • Specific examples of the alkali metal halide include lithium chloride and lithium bromide.
  • Specific examples of the alkaline earth metal halide include magnesium chloride and calcium chloride. Among these inorganic salts, lithium chloride and calcium chloride are particularly preferred.
  • the preparation of the dispersion may be easier.
  • the content of the inorganic salt is 0.1% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more based on the total amount of the dispersion. Or 15% by mass or more, and may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less.
  • the dispersion 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 modified fibroin in the spinning solution.
  • the modified spider silk fibroin used as a raw material is not particularly limited, and may be fibroin produced by a microorganism or the like by genetic recombination technology, or fibroin produced by synthesis. However, naturally derived spider silk fibroin is excluded from the modified spider silk fibroin.
  • modified spider silk fibroin refers to a spider silk fibroin having an amino acid sequence different from that of a naturally occurring spider silk fibroin. Spider silk fibroin having the same amino acid sequence as the spider silk fibroin.
  • spider silk fibroin of natural origin examples include spider silk fibroin produced by spiders such as large tubule wicking silk protein, weft silk protein, and small ampullate gland protein. Since the large spinneret thread has a repeating region including a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity.
  • the weft of spider silk has a feature that it does not have a crystalline region but has a repeating region composed of an amorphous region. The weft has a lower stress than the large spinneret and has a high elasticity.
  • the large spinal cord marker thread protein is produced by the large ampullate gland of spiders, and has the characteristic of excellent toughness.
  • Examples of the large spinal cord marker thread protein include the large ampullate spidroins MaSp1 and MaSp2 derived from the American spider (Nephila laclavipes), and ADF3 and ADF4 derived from Araneus diadematus.
  • ADF3 is one of the two major bookmarker thread proteins of the Japanese spider.
  • the spider silk 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.
  • spider silk fibroins produced by spiders include, for example, spiders belonging to the genus Araneus (Araneus sp.), Such as Orion spiders, Japanese spiders, A. spiders and A. spiders, and the spiders of the Japanese spiders Spiders belonging to the genus (Neoscona), spiders belonging to the genus Argiope (Pronus) such as Argiope serrata, spiders, spiders belonging to the genus Cyrarchachne such as the Torinofundamashi and Otorinofundamashi, and Spiders belonging to the genus Spider spiders (Genus Gasteracantha) such as Tibato spiders, spiders belonging to the genus Ordgarius, such as spiders belonging to the genus Orthodox spiders, such as the spider spider Spider spiders and the spiders spiders spiders, etc.
  • spiders belonging to the genus Araneus Such as Orion spiders, Japanese spiders, A. spiders and A. spiders,
  • Spiders belonging to the genus Argiope such as Argiope bruennichi and Argiope bruennichi, spiders belonging to the genus Arachnura (genus Arachnura) such as the arachnid spider, spiders such as the spiders belonging to the genus Acusilas and the spiders of the spider spiders belonging to the genus Acusilas such as the spider Spiders belonging to the genus Cytophora, spiders belonging to the spider spider belonging to the genus Cytophora (genus Poltys), spiders belonging to the genus Spiders belonging to the genus Spiders belonging to the genus Poltys, spiders belonging to the genus Spiders, spiders belonging to the spider, spiders belonging to the genus Cyclos sp.
  • Argiope Argiope
  • genus Arachnura genus Arachnura
  • spiders belonging to the genus Acusilas such as the arachnid
  • Spider silk proteins produced by spiders belonging to the genus Chorizopes, and spider silk spiders, Asagata spiders, Harabiroashida spiders, and urocore spiders The spiders belonging to the genus Tetragnatha (genus Tetragnatha), the spiders belonging to the genus Tetragnatha, the spiders belonging to the genus Leucauge, the spiders belonging to the genus Leucauge, and the genus E belonging to the spiders sp.
  • the spiders belonging to the genus L such as spiders belonging to the spiders belonging to the genus Menosira, such as the spider spider, the spiders belonging to the genus Dyschiriognatha, such as the spiders belonging to the genus Menosira, the spiders belonging to the spiders belonging to the spiders spiders belonging to the spiders, the black widow spider, the red widow spider, and the black spiders And spiders belonging to the genus Euprostenops (Tetragnathidae), such as spiders belonging to the genus Euprostenops Spider silk proteins produced by spiders.
  • spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ph ), U37520 (base sequence)), major ⁇ ampullate ⁇ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number
  • the modified spider silk fibroin according to the present embodiment is represented by, for example, Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It may be a protein containing a domain sequence.
  • an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to one or both of the N-terminal side and the C-terminal side of the domain sequence.
  • the N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.
  • a modified spider silk fibroin is preferably used as the modified fibroin because it is excellent in heat retention, moisture absorption and heat generation and / or flame retardancy.
  • domain sequence refers to a crystalline region unique to fibroin (typically, corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence).
  • the amino acid represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Means an array.
  • the (A) n motif indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues.
  • the number of amino acid residues in the n motif may be 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16.
  • the ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues).
  • At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • the REP may be an amino acid sequence composed of 10 to 200 amino acid residues, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160, or The amino acid sequence may be composed of 10 to 180 amino acid residues.
  • m represents an integer of 2 to 300, and 8 to 300, 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, It may be an integer of 20 to 140 or 20 to 120.
  • 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.
  • the modified spider silk fibroin is, for example, a modified spider silk fibroin whose amino acid sequence is modified based on the amino acid sequence of the spider silk fibroin (for example, by changing the gene sequence of a cloned naturally occurring spider silk fibroin,
  • the amino acid sequence may be modified or artificially designed and synthesized without using spider silk fibroin of natural origin (for example, a desired amino acid sequence can be synthesized by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). May be included).
  • the modified spider silk fibroin is, for example, an amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to the cloned gene sequence of naturally occurring spider silk fibroin. It can be obtained by making modifications. 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).
  • modified spider silk fibroin examples include a modified spider silk fibroin (first modified spider silk fibroin) derived from a large spinal canal thread protein produced in the large ampullate gland of a spider, and the content of glycine residues Spider silk fibroin (second modified spider silk fibroin), (A) modified spider silk fibroin with reduced n- motif content (third modified spider silk fibroin), glycine residue content And (A) a modified spider silk fibroin having a reduced content of the n motif (fourth modified spider silk fibroin), and a modified spider silk fibroin (fifth modified spider silk fibrin having a domain sequence including a region having a locally large hydrophobicity index). Modified spider silk fibroin) and a modified spider silk fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified silk fibroin). Yarn fibroin), and the like.
  • first modified spider silk fibroin derived from a large spinal canal thread protein produced in the large ampullate gland of
  • the modified spider silk fibroin (first modified spider silk fibroin) derived from the large spinal cord marker silk protein produced in the large ampullate of the spider is represented by Formula 1: [(A) n motif-REP] m And proteins containing the domain sequence to be performed.
  • n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, further preferably an integer of 8 to 20, and still more preferably an integer of 10 to 20.
  • An integer of 4 to 16 is still more preferred, an integer of 8 to 16 is particularly preferred, and an integer of 10 to 16 is most preferred.
  • the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. It is more preferably a residue, and even more preferably 20 to 75 residues.
  • the first modified spider silk fibroin has the total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m It is preferably at least 40%, more preferably at least 60%, even more preferably at least 70%, based on the total number of residues.
  • the first modified spider silk fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) n motif-REP] m and has a C-terminal sequence represented by any one of SEQ ID NOs: 1 to 3.
  • the protein may be a sequence or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOS: 1 to 3.
  • the amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is
  • the amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
  • the first modified spider silk fibroin (1-i) the amino acid sequence represented by SEQ ID NO: 4 or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 having 90% or more sequence identity Modified spider silk fibroin containing an amino acid sequence having sex properties.
  • the sequence identity is preferably 95% or more.
  • the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human ⁇ rhinovirus @ 3C protease) recognition site at the N-terminus is added.
  • the 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue.
  • the amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.
  • the modified spider silk fibroin of (1-i) may have an amino acid sequence represented by SEQ ID NO: 4.
  • the modified spider silk fibroin having a reduced content of glycine residues has a domain sequence in which the content of glycine residues is reduced as compared with a naturally occurring spider silk fibroin.
  • Having an amino acid sequence of The second modified spider silk fibroin has an amino acid sequence corresponding to at least one or more glycine residues in the REP has been replaced with another amino acid residue, as compared to a naturally occurring spider silk fibroin. It can be said.
  • the second modified spider silk fibroin has GGX and GPGXX in the REP (where G is a glycine residue, P is a proline residue, and X is other than glycine, as compared with the naturally occurring spider silk fibroin.
  • G is a glycine residue
  • P is a proline residue
  • X is other than glycine, as compared with the naturally occurring spider silk fibroin.
  • an amino acid sequence corresponding to the substitution of at least one or a plurality of glycine residues in the motif sequence with another amino acid residue is determined. You may have.
  • the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.
  • the second modified spider silk fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and from the above domain sequence, the (A) n motif located at the most C-terminal side to the above
  • the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence is represented by z
  • z / w is 30%
  • it may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
  • the second modified spider silk fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX below.
  • z / w (%) can be calculated by dividing z by w.
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70/70. %, Still more preferably 80% or more.
  • the upper limit of z / w is not particularly limited, but may be, for example, 95% or less.
  • the second modified spider silk fibroin is obtained, for example, by replacing at least a part of a nucleotide sequence encoding a glycine residue from a cloned naturally occurring spider silk fibroin gene sequence to encode another amino acid residue. It can be obtained by modification.
  • a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more.
  • an amino acid sequence satisfying the above aspect from the amino acid sequence of spider silk fibroin derived from nature, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues are further substituted,
  • the amino acid sequence corresponding to the deletion, insertion and / or addition may be modified.
  • the other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residues are more preferred, and glutamine (Q) residues are even more preferred.
  • a modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified spider silk fibroin of (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 6 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 10 corresponding to naturally occurring spider silk fibroin with GQX.
  • the amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In which one [(A) n motif-REP] was inserted.
  • the amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some of the N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7.
  • the amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating the above four times.
  • the value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 46.8%.
  • the values of z / w in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are 58.7%, respectively. 70.1%, 66.1% and 70.0%.
  • the value of x / y at the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 is as follows: They are 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.
  • the modified spider silk fibroin of (2-i) may have an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified spider silk fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified spider silk fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and is contained in REP.
  • the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) is z, and the total number of REP amino acids in the domain sequence is w, z / W is preferably at least 50.9%.
  • the second modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified spider silk fibroin can be isolated, immobilized, detected, visualized, and the like.
  • the tag sequence examples include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule.
  • affinity tag is a histidine tag (His tag).
  • His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography).
  • SEQ ID NO: 12 amino acid sequence including a His tag sequence and a hinge sequence.
  • tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can be used.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • an “epitope tag” utilizing an antigen-antibody reaction can be used.
  • a peptide (epitope) showing antigenicity as a tag sequence an antibody against the epitope can be bound.
  • the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag.
  • a tag sequence that can be cleaved by a specific protease can be used.
  • protease treatment By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified spider silk fibroin from which the tag sequence has been separated can also be recovered.
  • the second modified fibroin containing a tag sequence (2-iii) the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, or (2-iv) Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned.
  • amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively.
  • an amino acid sequence represented by SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 9.
  • the modified spider silk fibroin of (2-iii) may have an amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified spider silk fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified spider silk fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and is contained in REP.
  • the total number of amino acid residues in the amino acid sequence consisting of XGX is z, and the total number of REP amino acids in the domain sequence is w, z / W is preferably at least 50.9%.
  • the second modified spider silk fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • (A) n motifs modified spider silk fibroin content is reduced (Third modified spider silk fibroin), the domain sequence is compared to the naturally occurring silk fibroin, containing (A) n motif It has a reduced amino acid sequence. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) n motifs as compared to a naturally occurring spider silk fibroin.
  • the third modified spider silk fibroin may have an amino acid sequence corresponding to 10 to 40% deletion of the (A) n motif from a naturally occurring spider silk fibroin.
  • the third modified spider silk fibroin has a domain sequence of at least one for every 1-3 (A) n motifs from the N-terminus to the C-terminus compared to naturally occurring spider silk fibroin. A) It may have an amino acid sequence corresponding to the deletion of the n motif.
  • the third modified spider silk fibroin has a deletion of the (A) n motif whose domain sequence is at least two contiguous from the N-terminal side to the C-terminal side, as compared with the naturally-derived spider silk fibroin, and It may have an amino acid sequence corresponding to the deletion of one (A) n motif repeated in this order.
  • the third modified spider silk fibroin has an amino acid sequence whose domain sequence corresponds to the deletion of the (A) n motif at least every other region from the N-terminal to the C-terminal. Is also good.
  • the third modified spider silk fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) The number of amino acid residues of the REP of the [ n motif-REP] unit is sequentially compared, and when the number of amino acid residues of the REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.
  • the maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units of 8 to 11.3 is x, and the total number of amino acid residues in the domain sequence is y.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from spider silk fibroin. From the N-terminal side (left side), the domain sequence is (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called an n motif.
  • the number of amino acid residues of each REP in two selected adjacent [(A) n motif-REP] units is compared.
  • each pattern the total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
  • x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.
  • x / y is preferably at least 50%, more preferably at least 60%, further preferably at least 65%, and more preferably at least 70%. Even more preferably, it is even more preferably 75% or more, and particularly preferably 80% or more.
  • the upper limit of x / y is not particularly limited, and may be, for example, 100% or less.
  • x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y.
  • / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.
  • the third modified spider silk fibroin is a modified spider silk fibroin in which at least seven of the (A) n motifs present in a plurality in the domain sequence are composed of only alanine residues, x / y is 46.4% Or more, more preferably 50% or more, still more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, It is particularly preferred that it is 80% or more.
  • the upper limit of x / y is not particularly limited, and may be 100% or less.
  • the third modified spider silk fibroin is, for example, one or more of the sequences encoding the (A) n motif such that x / y is 64.2% or more from the cloned spider silk fibroin gene sequence. Can be obtained by deleting Further, for example, an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more from the amino acid sequence of spider silk fibroin derived from nature. Alternatively, it can be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or substituted.
  • the amino acid sequence corresponding to the addition may be modified.
  • the third modified spider silk fibroin (3-i) the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (3-ii) SEQ ID NO: 18 , A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified spider silk fibroin (3-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 18 is different from the amino acid sequence represented by SEQ ID NO: 10 corresponding to naturally occurring spider silk fibroin in that every two (A) n motifs are deleted from the N-terminal side to the C-terminal side. And one [(A) n motif-REP] was inserted in front of the C-terminal sequence.
  • the amino acid sequence represented by SEQ ID NO: 7 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 18 with GQX.
  • the amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some of the N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7.
  • the amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating the above four times.
  • the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8 to 11.3.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 18 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.3%.
  • the values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
  • the modified spider silk fibroin of (3-i) may have an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified spider silk fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified spider silk fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has C-terminal sequence from the N-terminal side.
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared toward the terminal side, when the number of amino acid residues of REP having a small number of amino acid residues is 1, Of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of the other REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3).
  • x / y be 64.2% or more, where x is the maximum value of the sum of the number of amino acid residues and y is the total number of amino acid residues in the domain sequence.
  • the third modified spider silk fibroin may include the above-described tag sequence at one or both of the N-terminus and the C-terminus.
  • the third modified spider silk fibroin including the tag sequence, (3-iii) the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (3-iv) ) Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
  • amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively.
  • an amino acid sequence represented by SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 9.
  • the modified spider silk fibroin of (3-iii) may have an amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified spider silk fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified spider silk fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and has C-terminal sequence from the N-terminal side.
  • the third modified spider silk fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • the modified spider silk fibroin (the fourth modified spider silk fibroin) in which the content of the glycine residue and the content of the (A) n motif are reduced has a domain sequence that is lower than that of a naturally derived spider silk fibroin.
  • (A) having an amino acid sequence in which the content of glycine residues is reduced in addition to the reduced content of the n motif.
  • the domain sequence of the fourth modified spider silk fibroin differs from the naturally occurring spider silk fibroin in that at least one or more (A) n motifs have been deleted, and at least one or more of the It can be said that the glycine residue has an amino acid sequence corresponding to the substitution of another amino acid residue.
  • the modified spider silk fibroin (the second modified spider silk fibroin) in which the content of the glycine residue was reduced and the content of the (A) n motif were reduced.
  • a modified spider silk fibroin having the characteristics of the modified spider silk fibroin (third modified spider silk fibroin). Specific embodiments and the like are as described for the second modified spider silk fibroin and the third modified spider silk fibroin.
  • the fourth modified spider silk fibroin (4-i) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, (4-ii) SEQ ID NO: 7, SEQ ID NO: 8 or Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9 can be mentioned.
  • Specific embodiments of the modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 are as described above.
  • the modified spider silk fibroin having a domain sequence including a region having a locally large hydrophobicity index has a domain sequence of 1 in REP compared to naturally occurring spider silk fibroin.
  • local substitution corresponding to substitution of a plurality of amino acid residues with amino acid residues having a large hydrophobicity index and / or insertion of one or more amino acid residues having a large hydrophobicity index into REP. May have an amino acid sequence containing a region having a large hydrophobicity index.
  • a region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
  • the fifth modified fibroin is that one or more amino acid residues in the REP have been replaced with amino acid residues having a large hydrophobicity index, and / or Or, in addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, further, compared with naturally occurring spider silk fibroin, substitution or deletion of one or more amino acid residues, insertion, And / or there may be an amino acid sequence modification corresponding to the addition.
  • the fifth modified spider silk fibroin is obtained by, for example, removing one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in REP from the cloned gene sequence of naturally occurring spider silk fibroin. It can be obtained by substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. Further, for example, one or more hydrophilic amino acid residues in REP are replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acids are contained in REP.
  • one or more hydrophilic amino acid residues in REP are replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acids are contained in REP.
  • amino acid sequence corresponding to insertion of a residue can also be obtained by designing an amino acid sequence corresponding to insertion of a residue and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of the naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acid residues in the REP.
  • the amino acid sequence may further be modified corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues.
  • the fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m and extends from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p,
  • q the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q
  • p / q is 6 .2% or more.
  • hydrophobicity index of amino acid residues
  • a publicly known index Kyte J, & Doolittle R (1982) "A simple method for display, the hydropathic charactor of aa protein, J.Pol.Mol. 105-132).
  • HI hydropathic index
  • sequence A [(A) n motif-REP] m (Hereinafter, referred to as “sequence A”).
  • sequence A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues).
  • the average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
  • p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Is still more preferred, and even more preferably 30% or more.
  • the upper limit of p / q is not particularly limited, but may be, for example, 45% or less.
  • the fifth modified spider silk fibroin is, for example, one or more hydrophilic amino acid residues in REP (for example, the amino acid sequence of the cloned naturally-derived spider silk fibroin is changed so as to satisfy the above-mentioned p / q conditions).
  • Replacing an amino acid residue with a negative hydrophobicity index with a hydrophobic amino acid residue eg, an amino acid residue with a positive hydrophobicity index
  • a hydrophobic amino acids in the REP It can be obtained by inserting a residue to locally modify the amino acid sequence to include a region having a large hydrophobicity index.
  • an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of spider silk fibroin derived from nature, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues in the REP were replaced with amino acid residues having a higher hydrophobicity index as compared to naturally occurring spider silk fibroin, and / or one or more amino acid residues in the REP.
  • the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues was performed. Is also good.
  • the amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
  • the fifth modified spider silk fibroin examples include (5-i) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (5-ii) SEQ ID NO: 19, SEQ ID NO: 20 or A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 21 can be mentioned.
  • the modified spider silk fibroin of (5-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 22 is obtained by deleting the amino acid sequence having consecutive alanine residues in the (A) n motif of a spider silk fibroin of natural origin so that the number of consecutive alanine residues becomes five. It is.
  • the amino acid sequence represented by SEQ ID NO: 19 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues at every other REP into the amino acid sequence represented by SEQ ID NO: 22; A part of the amino acids at the C-terminal side are deleted so that the molecular weight of the amino acid sequence to be obtained is almost the same.
  • the amino acid sequence represented by SEQ ID NO: 23 is different from the amino acid sequence represented by SEQ ID NO: 22 by inserting two alanine residues at the C-terminal side of each (A) n motif, and further including a part of glutamine (Q) residue.
  • a group is substituted with a serine (S) residue, and some amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence shown in SEQ ID NO: 22 is almost the same.
  • the amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 23.
  • the amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 23.
  • the modified spider silk fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
  • the modified spider silk fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most C-terminal (A )
  • the REP is included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more.
  • the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q. Sometimes, it is preferable that p / q is 6.2% or more.
  • the fifth modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus.
  • a modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 26.
  • amino acid sequences represented by SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 correspond to the amino acid sequence represented by SEQ ID NO: 12 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).
  • the modified spider silk fibroin of (5-iii) may have an amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
  • the modified spider silk fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
  • the modified spider silk fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located most C-terminal (A )
  • the REP is included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more.
  • the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q. Sometimes, it is preferable that p / q is 6.2% or more.
  • the fifth modified spider silk fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • the modified spider silk fibroin having a domain sequence with a reduced content of glutamine residues has a reduced content of glutamine residues as compared to naturally occurring spider silk fibroin Has an amino acid sequence.
  • the sixth modified spider silk fibroin preferably contains at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
  • the content of the GPGXX motif is usually 1% or more, and may be 5% or more, and preferably 10% or more.
  • the upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
  • the “GPGXX motif content” is a value calculated by the following method.
  • Formula 1 [(A) n motif -rep] m
  • Formula 2 [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal (A) In all REPs included in the sequence except for the sequence from the n motif to the C-terminus of the domain sequence from the n motif to the domain sequence, the total number of GPGXX motifs contained in the region was tripled ( That is, s is defined as s (corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence, and further (A) n Assuming that the total number of amino acid residues of all REPs excluding the motif is t, the GPGXX motif content is
  • “the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) sequence from the n motif to the C-terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of spider silk fibroin. Is small (that is, when the domain sequence is short), which affects the calculation result of the GPGXX motif content, so that this effect is eliminated.
  • “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “GPGXX motif”.
  • FIG. 3 is a schematic diagram showing the domain sequence of spider silk fibroin.
  • the method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of spider silk fibroin ("[(A) n motif-REP] m- (A) n motif" type) shown in FIG. 3, all REPs are located at the "most C-terminal position".
  • all REPs are “sequences in which the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence shown as “region A” in FIG. 3). ),
  • the sixth modified spider silk fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and more preferably 0%. Particularly preferred.
  • the “glutamine residue content” is a value calculated by the following method.
  • Formula 1 [(A) n motif -rep] m
  • Formula 2 [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs included in the sequence (sequence corresponding to “region A” in FIG.
  • the sixth modified spider silk fibroin had one or more glutamine residues in the REP deleted or substituted with other amino acid residues in the domain sequence compared to the naturally occurring spider silk fibroin. It may have an amino acid sequence corresponding to this.
  • the “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue.
  • the hydrophobicity index of amino acid residues is as shown in Table 1.
  • an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable.
  • the hydrophobicity of REP is preferably -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more. It is still more preferably 3 or more, and particularly preferably 0.4 or more.
  • the upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 0.7 or less.
  • REP hydrophobicity is a value calculated by the following method.
  • Formula 1 [(A) n motif -rep] m
  • Formula 2 [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs contained in the sequence (sequence corresponding to “region A” in FIG.
  • the sixth modified spider silk fibroin may have a domain sequence that lacks one or more glutamine residues in the REP, and / or one or more glutamine residues in the REP, as compared to the naturally occurring spider silk fibroin.
  • the modification corresponding to the substitution of a glutamine residue with another amino acid residue the modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues is also included. There may be.
  • the sixth modified spider silk fibroin may be, for example, by deleting one or more glutamine residues in the REP from the cloned naturally occurring spider silk fibroin gene sequence, and / or one or more glutamine in the REP. It can be obtained by replacing a residue with another amino acid residue.
  • one or more glutamine residues in REP have been deleted from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more glutamine residues in REP have been replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the substitution and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • the sixth modified spider silk fibroin (6-i) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: A modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 43, or (6-ii) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: Modified spider silk fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 43 can be mentioned.
  • the modified spider silk fibroin of (6-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 7 is based on the nucleotide sequence and amino acid sequence of a naturally occurring fibroin, Nephila clavipes (GenBank Accession Number: P468804.1, GI: 1174415), and (A) n
  • the amino acid sequence in which the alanine residues in the motif are consecutive has been modified such that the number of consecutive alanine residues is changed to 5, for example, to improve productivity.
  • the glutamine residue (Q) is not modified, so that the glutamine residue content is almost the same as that of naturally occurring fibroin.
  • M_PRT965 represented by SEQ ID NO: 28 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with TS and replacing the remaining Qs with A.
  • the amino acid sequence represented by SEQ ID NO: 29 (M_PRT889) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VL and replacing the remaining Qs with I.
  • the amino acid sequence (M_PRT525) represented by SEQ ID NO: 34 is obtained by inserting two alanine residues into a region (A 5 ) in which alanine residues are continuous with Met-PRT410 (SEQ ID NO: 7), In this example, two C-terminal domain sequences were deleted, and 13 glutamine residues (Q) were replaced with a serine residue (S) or a proline residue (P).
  • the amino acid sequence represented by SEQ ID NO: 43 (Met-PRT966) is obtained by converting all the QQs in the amino acid sequence represented by SEQ ID NO: 9 (the amino acid sequence before the amino acid sequence represented by SEQ ID NO: 42 is added to the C-terminus) to VF , And the remaining Q is replaced by I.
  • amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 43 all have a glutamine residue content of 9% or less. (Table 2).
  • the modified spider silk fibroin of (6-i) consists of the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 It may be something.
  • the modified spider silk fibroin of (6-ii) has an amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 and 90 % Or more.
  • the modified spider silk fibroin of (6-ii) is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Is a protein containing a domain sequence.
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified spider silk fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified spider silk fibroin can be isolated, immobilized, detected, visualized, and the like.
  • the sixth modified spider silk fibroin including the tag sequence, (6-iii) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: A modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 41 or SEQ ID NO: 44, or (6-iv) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 44 can be mentioned.
  • amino acid sequences represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44 are SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, respectively.
  • the amino acid sequence represented by SEQ ID NO: 12 (including the His tag sequence and the hinge sequence) was added to the N-terminal of the amino acid sequence represented by SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 43 Things.
  • amino acid sequence represented by SEQ ID NO: 44 has a glutamine residue content of 9% or less (Table 3).
  • the modified spider silk fibroin of (6-iii) consists of the amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44 It may be something.
  • the modified spider silk fibroin of (6-iv) has an amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44, and 90 % Or more.
  • the modified spider silk fibroin of (6-iv) is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Is a protein containing a domain sequence.
  • the sequence identity is preferably 95% or more.
  • the modified spider silk fibroin of (6-iv) preferably has a glutamine residue content of 9% or less.
  • the modified spider silk fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified spider silk fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • the modified spider silk fibroin comprises a first modified spider silk fibroin, a second modified spider silk fibroin, a third modified spider silk fibroin, a fourth modified spider silk fibroin, a fifth modified spider silk fibroin, and a sixth modified spider silk fibroin.
  • the modified spider silk fibroin having at least two or more characteristics may be used.
  • the modified spider silk fibroin may be a hydrophilic modified spider silk fibroin or a hydrophobic modified spider silk fibroin.
  • Hydrophobic modified spider silk fibroin refers to a value obtained by calculating the sum of the hydrophobicity indexes (HI) of all amino acid residues constituting the modified spider silk fibroin, and then dividing the sum by the total number of amino acid residues (average HI). ) Is 0 or more.
  • the hydrophobicity index is as shown in Table 1.
  • the hydrophilic modified spider silk fibroin is a modified spider silk fibroin having the above average HI of less than 0.
  • hydrophobic modified spider silk fibroin examples include the sixth modified fibroin described above. More specific examples of the hydrophobically modified spider silk fibroin include amino acids represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 Modified spider silk fibroin comprising the amino acid sequence represented by the sequence, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44.
  • hydrophilic modified spider silk fibroin examples include the above-mentioned first modified fibroin, second modified fibroin, third modified fibroin, fourth modified fibroin, and fifth modified fibroin.
  • More specific examples of the hydrophilic spider silk protein include an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: Modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the above-mentioned modified spider silk fibroin can be used alone or in combination of two or more.
  • the modified spider silk fibroin is, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified spider silk fibroin 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 modified spider silk fibroin is not particularly limited.
  • the nucleic acid can be produced by a method of amplifying and cloning by polymerase chain reaction (PCR) using a gene encoding a modified spider silk fibroin, 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 spider silk proteins obtained from the NCBI web database or the like, AKTA oligopilot plus 10/100 (GE Healthcare Japan KK) Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like by PCR or the like.
  • a nucleic acid encoding the modified spider silk fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus was synthesized. You may.
  • the regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host.
  • An inducible promoter that functions in a host cell and is capable of inducing the expression of the desired modified spider silk fibroin 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 modified spider silk fibroin 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.
  • the expression vector is capable of autonomous replication in the prokaryote and contains a promoter, a ribosome binding sequence, a nucleic acid encoding a modified spider silk fibroin, and a transcription termination sequence. It is preferably a vector. A gene that controls the promoter may be included.
  • Prokaryotes include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like.
  • Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli.
  • Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri.
  • Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like.
  • 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.
  • a prokaryote when used as a host, as a vector for introducing a nucleic acid encoding a modified spider silk fibroin, 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.
  • pBTrp2 manufactured by Boehringer Mannheim
  • pGEX manufactured by Pharmacia
  • pUC18 pBluescriptII, pSuex, pET22b, pCold
  • pUB110 pNCO2
  • 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 modified spider silk fibroin is introduced include YEp13 (ATCC 37115) and YEp24 (ATCC 37051).
  • any 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 modified spider silk fibroin can be produced, for example, by culturing the transformed host in a culture medium, producing and accumulating the modified spider silk fibroin in the culture medium, and collecting the modified spider silk fibroin from the culture medium.
  • the method of culturing the transformed host in a culture medium can be performed according to a method generally used for culturing a host.
  • the culture medium contains a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, thereby efficiently culturing the host.
  • a natural medium or a synthetic medium may be used as long as the medium can be used.
  • the carbon source may be any as long as the host can assimilate it.
  • examples include glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysates, and organic acids such as acetic acid and propionic acid. And alcohols such as ethanol and propanol.
  • 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.
  • 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.
  • potassium (I) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate
  • potassium (I) phosphate potassium (II) phosphate
  • magnesium phosphate magnesium phosphate
  • magnesium sulfate sodium chloride
  • ferrous sulfate manganese sulfate
  • copper sulfate copper sulfate
  • calcium carbonate calcium carbonate
  • ⁇ 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 during the culture.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside or the like is used.
  • An acid or the like may be added to the medium.
  • the modified spider silk fibroin produced by the transformed host can be isolated and purified by a method usually used for protein isolation and purification.
  • a method usually used for protein isolation and purification For example, when the modified spider silk fibroin is expressed in a dissolved state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, The host cells are crushed with a Manton-Gaurin homogenizer, Dynomill or the like 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).
  • the modified spider silk fibroin when expressed by forming an insoluble body in the cells, the host cells are similarly recovered, crushed, and centrifuged to obtain an insoluble body of the modified spider silk fibroin as a precipitate fraction. Collect.
  • the recovered insoluble form of the modified spider silk fibroin can be solubilized with a protein denaturant. After this operation, a purified sample of the modified spider silk fibroin can be obtained by the same isolation and purification method as described above.
  • the modified spider silk fibroin When the modified spider silk fibroin is secreted extracellularly, the modified spider silk fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the method for producing a modified spider silk fibroin fiber includes a step of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming a raw fiber from the dispersion. And at least a step.
  • fibrils refers to fibrous solids formed from a dispersion used as a spinning dope (dope) by cooling the dispersion, evaporating a solvent (vaporizing), a chemical reaction, or the like.
  • the step of forming fibrils from the dispersion may include discharging the above-described dispersion, or may include drawing the fibrils from the above-described dispersion, discharging the dispersion, and It may include both drawing fibrils from the dispersion.
  • the fibrils formed from the dispersion may be used as modified spider silk fibroin fibers as they are, or modified spider silk fibroin fibers obtained by subjecting the fibrils to processing such as stretching described below. Good.
  • the method for producing a modified spider silk fibroin fiber according to the present embodiment may further include a step of defoaming a dispersion used as a spinning stock solution (dope solution) (defoaming step).
  • the liquid When performed in a liquid, the liquid is preferably a liquid having a low affinity for the dispersion, for example, a non-polar solvent.
  • the point of contact between the dispersion and the device When drawing fibrils is performed in a gas, the point of contact between the dispersion and the device may be at the gas / dispersion interface, and when drawing fibrils in a liquid, The point of contact between the dispersion and the device may be at the liquid / dispersion interface. By drawing the fibrils, the fiber diameter of the fibrils can be made smaller.
  • the device to be brought into contact with the dispersion is preferably one having a sharp tip in order to form a small point of contact with the dispersion, and examples thereof include a needle, a pipette tip, tweezers, a skewer, a toothpick, and a thin stick.
  • examples of the material of the device include, but are not limited to, plastic, metal, glass, and wood.
  • the speed at which the device is withdrawn in the direction away from the point of contact with the dispersion is adjusted by the viscosity of the dispersion. Further, it is preferable that the drawing speed is constant. By keeping the pulling speed constant, it is possible to prevent the fiber from being twisted, sagged or cut, and to suppress the variation in the fiber diameter.
  • the drawing speed for example, 0.1 cm / sec or more, 0.1 cm / sec to 15 m / sec, 1 cm / sec to 5 m / sec, 3 cm / sec to 1 m / sec, 5 cm / sec to 50 cm / sec, 5 cm / sec 3535 cm / sec.
  • the tip of the fibril and the tip of the instrument are adhered, but once the fibril is pulled out, the tip of the instrument and the Does not have to be bonded to the tip.
  • the fibrils continue to be drawn out of the dispersion liquid even if the tip of the device and the tips of the fibrils are not bonded.
  • discharge of dispersion liquid means discharging the dispersion liquid from the nozzle with or without applying pressure to the dispersion liquid.
  • the fine fibers are formed by the dispersion liquid finely discharged through the nozzle becoming solid by evaporation (vaporization) of the solvent, chemical reaction, or the like.
  • the destination where the dispersion liquid is discharged may be discharged into the air or may be discharged into a liquid (coagulating liquid).
  • the method of discharging the dispersion liquid from the nozzle is not particularly limited.
  • a method using a metering pump as a means for sending the spinning solution can be used.
  • the discharge amount can be appropriately adjusted according to the production speed.
  • a spinneret may be used as a nozzle for discharging the dispersion.
  • the spinneret shape, hole shape, number of holes, and the like of the spinneret are not particularly limited, and can be appropriately selected according to the desired fiber diameter, the number of single yarns, and the like.
  • the hole diameter of the spinneret can be, for example, 0.01 mm or more and 0.6 mm or less.
  • the hole diameter is 0.01 mm or more, the pressure loss can be reduced and the equipment cost can be reduced.
  • the pore diameter is 0.6 mm or less, it is possible to reduce the necessity of a stretching operation for reducing the fiber diameter, and it is possible to reduce the possibility of stretching breakage from discharge to winding.
  • either the discharging of the dispersion or the extraction of the fibrils may be performed to form the fibrils, or both may be performed.
  • the dispersion is discharged from a nozzle, the tip of the device is brought into contact with the discharged dispersion, and the device is dispersed. By pulling out the fiber in a direction away from the fiber and pulling out the fiber from the contact point, the fiber can be formed.
  • the dry spinning method is a method of spinning (forming fibrils) by evaporating (vaporizing) a solvent in a spinning solution (dope solution).
  • a spinning solution a spinning solution
  • the drawing of the fibrils from the dispersion may be performed in a gas such as air.
  • the discharge may be performed in a gas such as air.
  • the fibrils are formed by evaporating the solvent in the dispersion. That is, in the present embodiment, in the step of forming the fibrils, the fibrils may be formed by a dry spinning method.
  • Examples of the method for evaporating the solvent include drying methods used in known dry spinning methods such as hot air drying, heat drying, air drying, and natural drying.
  • drying methods used in known dry spinning methods such as hot air drying, heat drying, air drying, and natural drying.
  • the solvent evaporates spontaneously without performing aggressive drying such as hot-air drying, heat-drying, and air-drying, and the fibrils are removed. It can be formed and is efficient.
  • carbon black nanoparticles are used as the carbon material contained in the dispersion, it is preferable to form the fibrils by a dry spinning method.
  • the formed fibrils may be wound by a winding device such as a winder.
  • a winding device By using a winding device, fibers can be continuously produced. Further, after the raw fiber is drawn out, the raw fiber can be continuously drawn out by winding the raw fiber with a winding device.
  • a winder may be used as the winding device. In the winder, winding can be performed by appropriately adjusting winding conditions such as tension and contact pressure. As the winder, a known winder may be used.
  • the fiber diameter of the fibrils can be controlled. Specifically, the fiber diameter can be made thinner by increasing the winding speed, and a fiber having a large fiber diameter can be manufactured by reducing the winding speed.
  • the method for producing the modified spider silk fibroin fiber may further include a step of drawing the formed raw fiber (drawing step).
  • the original fiber is drawn at an arbitrary draw ratio, and the drawn fiber can be used as a modified spider yarn fibroin fiber of the present embodiment for any use.
  • Examples of the stretching method include wet heat stretching and dry heat stretching.
  • Wet heat stretching can be performed in warm water, a solution obtained by adding an organic solvent or the like to warm water, or steam heating.
  • the wet heat stretching temperature is preferably from 50 to 90 ° C, more preferably from 75 to 85 ° C.
  • the temperature is 50 ° C. or more, the pore diameter in the fiber can be made small and stable.
  • the temperature is 90 ° C. or lower, the temperature can be easily set and spinning stability is improved.
  • the draw ratio in the wet heat drawing is, for example, 1 to 30 times, 1 to 25 times, 1 to 20 times, or 1 to 30 times with respect to the undrawn yarn (or pre-drawn yarn). 1515 times, 1 ⁇ 10 times, 2 ⁇ 10 times, 2 ⁇ 8 times, 2 ⁇ 6 times, 2 ⁇ 24 times And may be 2-3 times.
  • Dry heat drawing can be performed by drawing in the air using a device equipped with a heat source such as a contact hot plate and a non-contact furnace, but the device is not particularly limited. Any device can be used as long as it can raise the temperature of the fiber to a predetermined temperature and can draw the fiber at a predetermined magnification.
  • the temperature at which the dry heat stretching is performed may be, for example, 100 ° C to 270 ° C, 140 ° C to 230 ° C, 140 ° C to 200 ° C, or 160 ° C to 200 ° C. , 160 ° C to 180 ° C.
  • the draw ratio in the dry heat drawing step may be, for example, 1 to 30 times, 1 to 25 times, or 1 to 20 times with respect to the undrawn yarn (or pre-drawn yarn). 1 to 15 times, 1 to 10 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, 2 to 4 times And may be 2-3 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, and the second-stage stretching is performed by wet-heat stretching. Stretching can be performed by appropriately combining wet heat stretching and dry heat stretching, such as by performing dry heat stretching.
  • the lower limit of the final draw ratio of the fiber after the drawing step is, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or more with respect to the undrawn yarn (or pre-drawn yarn). It may be any of double, eight, or nine times.
  • the upper limit of the final draw ratio of the fiber after the drawing step is, for example, any one 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 draw ratio is not limited as long as the desired properties such as fiber thickness and mechanical properties can be obtained.
  • an oil agent may be added to the fiber, if necessary, for the purpose of imparting antistatic properties, convergence properties, lubricity and the like.
  • the type of oil agent to be applied, the amount to be applied, and the like are not particularly limited, and can be appropriately adjusted in consideration of the use of the fiber, the handleability of the fiber, and the like.
  • the modified spider silk fibroin fiber according to the present embodiment can be manufactured.
  • the above-described method is an example, and the modified spider silk fibroin fiber according to the present embodiment may be manufactured by a method other than the above-described method.
  • the modified spider silk fibroin fiber according to the present embodiment is a fiber containing the modified spider silk fibroin and one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles.
  • the content of the carbon material may be from 0.01 part by mass to 1 part by mass, from 0.01 part by mass to less than 1 part by mass, based on the content of the modified spider silk fibroin.
  • 03 parts by mass or more and less than 1 part by mass may be 0.05 parts by mass or more and less than 1 part by mass, may be 0.05 parts by mass or more and 0.95 parts by mass or less, and may be 0.05 parts by mass.
  • It may be 0.9 parts by mass or less, may be 0.05 parts by mass or more and 0.8 parts by mass or less, may be 0.05 parts by mass or more and 0.7 parts by mass or less, and may be 0.05 parts by mass or less. It may be not less than 0.6 part by mass and not less than 0.05 part by mass, and may be not less than 0.05 part by mass and not more than 0.3 part by mass.
  • the method for producing a modified spider silk fibroin nonwoven fabric includes the steps of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming fibrils from the dispersion. And at least a step of intertwining the fibrils or modified spider silk fibroin fibers produced from the fibrils.
  • the manufactured modified spider silk fibroin nonwoven fabric may be used for any purpose as it is.
  • the step of forming the fibrils may include discharging the dispersion described above, and may include extracting the fibrils from the dispersion described above, and discharging the dispersion and from the dispersion. It may include both drawing fibrils.
  • the modified spider silk fibroin nonwoven fabric according to the present embodiment may be manufactured by a known nonwoven fabric manufacturing method using raw fibers or modified spider silk fibroin fibers manufactured from raw fibers.
  • a web including a single-layer web and a laminated web
  • a chemical bond method The fibers of the web can be bonded together by a dipping method, a spraying method, etc.
  • a needle punching method to obtain a nonwoven fabric.
  • the manufacturing process of the nonwoven fabric may further include a process of adjusting the fiber density of the nonwoven fabric (adjustment process).
  • the fiber density (basis weight) is a value defined by the weight per unit area of the nonwoven fabric.
  • the fiber density of the nonwoven fabric can be adjusted by, for example, increasing or decreasing the amount of fibers constituting the web, and in the case of a laminated web, can be adjusted by increasing or decreasing the number of layers.
  • Various products including the modified spider silk fibroin fiber according to the present embodiment can be manufactured.
  • Such products include, for example, fabrics such as fibers, yarns, and fabrics, knits, braids, non-woven fabrics, paper, and cotton.
  • the fiber include a long fiber, a short fiber, a monofilament, and a multifilament
  • examples of the yarn include a spun yarn, a twisted yarn, a false twisted yarn, a processed yarn, a mixed fiber, and a mixed spun yarn.
  • fabrics such as woven fabrics, knits, braids, non-woven fabrics, and the like, paper, cotton, and the like can be manufactured. These products can be manufactured by a known method.
  • the present invention also provides a method for dispersing a carbon material, comprising a step of mixing a modified spider silk fibroin, a carbon material of 25 parts by mass or less based on 100 parts by weight of the modified spider silk fibroin, and a polar solvent.
  • the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles.
  • the present invention also provides a dispersion aid for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin.
  • a dispersion aid for dispersing a carbon material in a polar solvent comprising a modified spider silk fibroin.
  • the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles.
  • the modified spider silk fibroin improves the dispersibility of the carbon material in the polar solvent. That is, the modified spider silk fibroin can be used as a dispersion aid for the carbon material.
  • the present invention also provides a thinning agent for producing a reduced modified spider silk fibroin fiber, comprising one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles.
  • a thinning agent for producing a reduced modified spider silk fibroin fiber, comprising one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles.
  • the specific carbon material can have an extremely small fiber diameter. That is, the specific carbon material can be used as a diameter reducing agent for producing a modified spider silk fibroin fiber having a reduced diameter.
  • the amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues with respect to the amino acid sequence of fibroin derived from Nephila clavipes for the purpose of improving productivity. Further, an amino acid sequence represented by SEQ ID NO: 12 (tag sequence and hinge sequence) is added to the N-terminus.
  • nucleic acids encoding the designed spider silk fibroins PRT799, PRT918, and PRT966 having the designed amino acid sequences represented by SEQ ID NO: 15, SEQ ID NO: 39, and SEQ ID NO: 44 were synthesized.
  • An NdeI site at the 5 'end and an EcoRI site downstream of the stop codon were added to the nucleic acid.
  • the nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • the seed culture solution was added to a jar fermenter to which 500 mL of a production medium (Table 5) had been added so that the OD 600 was 0.05.
  • the temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, the concentration of dissolved oxygen in the culture solution 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. Further, the culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the modified spider silk fibroin.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the precipitate after washing is suspended in 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, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after the dialysis was collected by centrifugation, the water was removed with a freeze dryer, and the freeze-dried powder was collected to obtain modified spider silk fibroin (PRT799, PRT918, PRT966).
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • Example 1 (Preparation of dispersion liquid) (Example 1) 3.16 g of formic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide (manufactured by Sigma-Aldrich, product number: 796034) were mixed, and the temperature was 60 ° C. using an ultrasonic cleaning device (manufactured by BRANSON). For 100 minutes by ultrasonic vibration. Immediately, 1 g of the modified spider silk fibroin (PRT799) obtained in the above-described production step was added, and the mixture was heated and dissolved with an aluminum block heater at 40 ° C. for 1 hour while stirring to prepare a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
  • Example 2 A dispersion was prepared in the same manner as in Example 1, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 0.5% by mass. did. Specifically, the amount of formic acid added was 1.9 g, the amount of modified spider silk fibroin was 0.1 g, and the amount of graphene oxide was 0.5 mg.
  • Example 3 A dispersion was prepared in the same manner as in Example 1, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 1% by mass.
  • Example 4 3.16 g of dimethyl sulfoxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide were mixed, and stirred by ultrasonic vibration at a temperature of 60 ° C. for 100 minutes using an ultrasonic cleaning device. Immediately, 1 g of the modified fibroin (PRT799) obtained in the process of producing the modified fibroin was added, and the mixture was heated with a 90 ° C. aluminum block heater for 3 hours to dissolve with stirring, thereby preparing a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
  • Example 5 A dispersion was prepared in the same manner as in Example 4, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 4% by mass.
  • Example 6 A dispersion was prepared in the same manner as in Example 4, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 20% by mass.
  • Comparative Example 1 As a comparative example, the dispersibility of graphene oxide in a formic acid single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 1, except that the modified spider silk fibroin was not added.
  • Comparative Example 2 As a comparative example, the dispersibility of graphene oxide in a DMSO single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 4, except that the modified spider silk fibroin was not added.
  • the dispersibility of graphene oxide in the dispersion was evaluated.
  • the dispersibility was evaluated by visually confirming the precipitate of graphene oxide in the dispersion liquid after stirring.
  • Table 6 shows the evaluation results of the dispersibility.
  • the evaluation criteria for dispersibility are as follows. :: After the stirring, the dispersion state is maintained even after being left for one month or more. :: After stirring, the dispersion state is maintained even if the mixture is allowed to stand for 14 days or more. ⁇ : sedimented within 48 hours after stirring. ⁇ : sedimentation within 24 hours after stirring.
  • Example 7 Preparation of dope solution (spinning stock solution) 3.16 g of formic acid and 1 mg of graphene oxide were mixed, and the mixture was stirred by ultrasonic vibration at a temperature of 60 ° C. for 99 minutes using an ultrasonic cleaning device, and a graphene oxide / formic acid dispersion was obtained.
  • a graphene oxide / formic acid dispersion was obtained.
  • 1 g of the modified spider silk fibroin (PRT799) obtained in the above-mentioned modified fibroin production step was added to the graphene oxide / formic acid dispersion, and heated with a 40 ° C aluminum block heater for 2 hours with stirring to dissolve. And a dope liquid (dispersion liquid) was prepared.
  • the obtained dope liquid was a viscous black liquid and had a viscosity at 25 ° C. of 6,230 [mPa ⁇ sec].
  • the added amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dope solution.
  • the addition amount of graphene oxide was 0.1% by mass based on the modified fibroin.
  • (2) Dry spinning Dry spinning was performed at room temperature using a spinning apparatus.
  • the prepared graphene oxide-containing dope liquid (dispersion liquid) was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle.
  • the tip of the needle was brought into contact with the end of the droplet to draw out the dope solution into a fibrous form.
  • the drawn fiber was wound up by a winder to form a fiber continuously while drafting, thereby obtaining a modified spider silk fibroin fiber.
  • the winding speed of the winder was set to the maximum speed of 20.82 m / min (34.7 cm / sec).
  • FIG. 4 shows an SEM image of the modified spider silk fibroin fiber obtained in Example 7.
  • Table 7 shows the analysis result (fiber diameter) of the SEM image of FIG.
  • the ImageJ software is described in the following document. Schindelin, J .; Arganda-Carreras, I .; & France, E.C. et al.
  • DiameterJ plug-in is described in the following document. Hotaling NA, Bharti K, Kriel H, Simon Jr. CG. DiameterJ: Validated open source nanofiber diameter measurement tool. Biomaterials 2015; 61: p. 327-38. doi: 10.1016 / j. biomaterials. 2015.05.015.
  • a dope solution containing no graphene oxide was prepared.
  • a dope solution was prepared in the same procedure as in Example 7 except that graphene oxide was not added.
  • (2) Dry spinning Dry spinning was performed at room temperature using a tabletop spinning apparatus in the same manner as in Example 7.
  • the prepared dope solution was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle.
  • the tip of the needle When a droplet was formed at the tip of the nozzle, the tip of the needle was brought into contact with the end of the droplet, and an attempt was made to draw out the dope solution in a fibrous form. In addition, the dope solution could not be drawn out into a fibrous form, and fibers could not be formed.
  • Example 8 (Production and evaluation of nonwoven fabric) (Example 8) (1) Preparation of Dope Solution (Spinning Stock Solution) In the same manner as in Example 3, a dope solution of graphene oxide / formic acid / modified spider silk fibroin (PRT799) was prepared. (2) Dry spinning Dry spinning was performed using a commercially available cotton candy manufacturing apparatus (manufactured by Azuma Co., Ltd.). The apparatus was turned on, and the disk-shaped rotating section inside the pan of the cotton candy manufacturing apparatus was rotated. The disk-shaped rotating part is a rotating part around an inlet for feeding a rough or the like when a cotton candy is manufactured using a cotton candy confectionery apparatus as usual.
  • the prepared graphene oxide-containing dope solution was filled in a syringe, and defoamed by centrifugation.
  • a dope solution is discharged into the air from a 0.2-mm-diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet contacts the disk-shaped rotating part of the cotton candy manufacturing device.
  • the fibers were continuously formed by utilizing the rotation of the disk-shaped rotating portion to obtain a modified spider silk fibroin nonwoven fabric.
  • FIG. 5 shows an SEM image of the nonwoven fabric obtained in Example 8.
  • Table 8 shows the analysis result of the SEM image of FIG. (Comparative Example 4)
  • (1) Preparation of Dope Solution As a comparative example, a dope solution containing no graphene oxide was prepared. A dope solution was prepared in the same procedure as in Example 8, except that graphene oxide was not added. (2) Dry spinning The prepared dope solution was filled in a syringe and defoamed by centrifugation.
  • the dry spinning was performed using a commercially available cotton candy manufacturing apparatus in the same manner as in Example 2.
  • the apparatus was turned on and the rotating pan was rotated.
  • the dope liquid is discharged into the air from a 0.2 mm diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet is brought into contact with a disk-shaped rotating portion of a cotton candy manufacturing apparatus. An attempt was made to form fibers, but no fibers could be formed.
  • the dope solution to which no graphene oxide was added failed to form a nonwoven fabric by dry spinning, whereas the dope solution to which graphene oxide was added (Example 8).
  • a nonwoven fabric could be formed by dry spinning.
  • the average fiber diameter of the obtained modified spider silk fibroin nonwoven fabric was as small as 1 to 2 ⁇ m, and unexpectedly excellent results could be obtained.
  • the nonwoven fabric had a sufficiently small fiber diameter, a heating step for removing (vaporizing) the solvent from the dope solution was unnecessary.
  • Reference Example 1 Flammability test of modified fibroin A freeze-dried powder of modified fibroin (PRT799) was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was added. Used and mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
  • the obtained spinning stock solution was heated to 90 ° C., filtered through a metal filter having a mesh size of 5 ⁇ m, and allowed to stand in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 90 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
  • a knitted fabric (thickness: 180 denier, gauge number: 18) was manufactured by circular knitting using a circular knitting machine by using a twisted yarn obtained by twisting raw fibers. 20 g of the obtained knitted fabric was cut out and used as a test piece.
  • the flammability test was based on the “Test method for synthetic resin with powdery or low melting point” described in “Fire Danger No. 50 (May 31, 1995)”. The test was performed under the conditions of a temperature of 22 ° C., a relative humidity of 45%, and an air pressure of 1021 hPa. Table 9 shows the measurement results (oxygen concentration (%), burning rate (%), reduced burning rate (%)).
  • the knitted fabric made of the modified fibroin (PRT799) fiber had a limiting oxygen index (LOI) value of 27.2.
  • LOI limiting oxygen index
  • the LOI value is 26 or more, it is known to be flame retardant. It can be seen that the modified fibroin has excellent flame retardancy.
  • Reference Example 2 Evaluation of heat generation by moisture absorption of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethyl sulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was used. And mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
  • the obtained spinning dope was heated to 60 ° C., filtered with a metal filter having a mesh size of 5 ⁇ m, and then allowed to stand in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
  • Table 10 shows the thickness and gauge number of the knitted fabric using PRT918 fiber or PRT799 fiber.
  • the thickness and the number of gauges of the knitted fabric using other raw material fibers were adjusted so that the same cover factor as the knitted fabric of the modified fibroin fiber was obtained. Specifically, it is as follows.
  • test piece Two knitted fabrics cut to 10 cm ⁇ 10 cm were aligned, and four sides were sewn to obtain a test piece (sample). After leaving the test specimen in a low humidity environment (temperature 20 ⁇ 2 ° C, relative humidity 40 ⁇ 5%) for 4 hours or more, it was moved to a high humidity environment (temperature 20 ⁇ 2 ° C, relative humidity 90 ⁇ 5%) The temperature was measured at 1 minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.
  • FIG. 6 is a graph showing an example of the result of the moisture absorption / heating test.
  • the horizontal axis of the graph represents the time (minute) of leaving the sample in the high humidity environment as 0 when the time when the sample was transferred from the low humidity environment to the high humidity environment.
  • the vertical axis of the graph indicates the temperature (sample temperature) measured by the temperature sensor.
  • the point indicated by M corresponds to the maximum value of the sample temperature.
  • Table 11 shows the calculation results of the maximum heat of moisture absorption of each knitted fabric.
  • the modified fibroin (PRT918 and PRT799) has a higher maximum heat of moisture absorption and excellent heat generation by moisture absorption than the existing materials.
  • Reference Example 3 Evaluation of heat retention of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and the mixture was shaken using a shaker. The mixture was dissolved by mixing for 3 hours. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
  • the obtained spinning dope was heated to 60 ° C., filtered through a metal filter having a mesh size of 5 ⁇ m, and then left standing in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
  • a knitted fabric was produced by flat knitting using a flat knitting machine.
  • Table 12 shows the count, the number of twists, the number of gauges, and the basis weight of the knitted fabric using the PRT966 fiber or the PRT799 fiber.
  • the knitted fabric using other raw material fibers was adjusted to have almost the same cover factor as the knitted fabric of the modified fibroin fiber. Specifically, it is as follows.
  • the heat retention was evaluated using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd., using a dry contact method (a method assuming that the skin and clothing directly touched in a dry state).
  • a dry contact method a method assuming that the skin and clothing directly touched in a dry state.
  • One knitted fabric cut into a rectangle of 20 cm ⁇ 20 cm was used as a test piece (sample).
  • the test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) radiated through the test piece was determined under the condition of a wind speed in the wind tunnel of 30 cm / sec. Without setting the test piece, the amount of heat (b) radiated under the same conditions as above was determined, and the heat retention (%) was calculated according to the following formula B.
  • Insulation index Insulation rate (%) / Sample weight (g / m 2 )
  • Table 11 shows the calculation results of the heat retention index. The higher the heat retention index, the more the material can be evaluated as having excellent heat retention.
  • the modified fibroin (PRT966 and PRT799) has a high heat retention index and is excellent in heat retention as compared with existing materials.
  • the modified fibroin when the modified fibroin is modified spider silk fibroin, the heat retention, the moisture absorption and heat generation and / or the flame retardancy can be more excellent.
  • the modified spider silk fibroin By using the modified spider silk fibroin to produce the fiber of the present invention, a fiber having excellent heat retention, moisture absorption and heat generation and / or flame retardancy, and a small diameter can be obtained.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Textile Engineering (AREA)
  • Artificial Filaments (AREA)
  • Processes Of Treating Macromolecular Substances (AREA)
  • Compositions Of Macromolecular Compounds (AREA)

Abstract

La présente invention concerne une dispersion de matériau carboné contenant une fibroïne de soie d'araignée modifiée, un matériau carboné, et un solvant polaire, le matériau carboné étant un graphène plan composé d'une ou plusieurs couches.
PCT/JP2019/038375 2018-09-28 2019-09-27 Fibre de fibroïne de soie d'araignée modifiée et son procédé de production Ceased WO2020067514A1 (fr)

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JP2018185291A JP2022024193A (ja) 2018-09-28 2018-09-28 改変クモ糸フィブロイン繊維及びその製造方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113018511A (zh) * 2021-02-25 2021-06-25 淮阴工学院 一种矿化纤维增强三维多孔支架及其制备方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014002605A1 (fr) * 2012-06-28 2014-01-03 スパイバー株式会社 Fibre de protéine teintée dans la masse et procédé pour produire celle-ci
CN108452374A (zh) * 2018-05-09 2018-08-28 海南蛛王药业有限公司 一种蜘蛛丝组合物及其制备方法
WO2018164234A1 (fr) * 2017-03-10 2018-09-13 カジナイロン株式会社 Procédé de production de fibre de protéine et procédé de rétraction de fibre de protéine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014002605A1 (fr) * 2012-06-28 2014-01-03 スパイバー株式会社 Fibre de protéine teintée dans la masse et procédé pour produire celle-ci
WO2018164234A1 (fr) * 2017-03-10 2018-09-13 カジナイロン株式会社 Procédé de production de fibre de protéine et procédé de rétraction de fibre de protéine
CN108452374A (zh) * 2018-05-09 2018-08-28 海南蛛王药业有限公司 一种蜘蛛丝组合物及其制备方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113018511A (zh) * 2021-02-25 2021-06-25 淮阴工学院 一种矿化纤维增强三维多孔支架及其制备方法与应用

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