WO2020158900A1 - Procédé de production de fibres de cheveux synthétiques, procédé de production de cheveux synthétiques, fibres de cheveux synthétiques et cheveux synthétiques - Google Patents

Procédé de production de fibres de cheveux synthétiques, procédé de production de cheveux synthétiques, fibres de cheveux synthétiques et cheveux synthétiques Download PDF

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WO2020158900A1
WO2020158900A1 PCT/JP2020/003551 JP2020003551W WO2020158900A1 WO 2020158900 A1 WO2020158900 A1 WO 2020158900A1 JP 2020003551 W JP2020003551 W JP 2020003551W WO 2020158900 A1 WO2020158900 A1 WO 2020158900A1
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fibroin
amino acid
seq
fiber
sequence
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PCT/JP2020/003551
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Japanese (ja)
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正敏 関
高橋　英樹
廉 伊藤
佑之介 安部
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株式会社アデランス
Ｓｐｉｂｅｒ株式会社
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    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41G—ARTIFICIAL FLOWERS; WIGS; MASKS; FEATHERS
    • A41G3/00—Wigs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/04—Preparations for permanent waving or straightening the hair
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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 method for producing a fiber for artificial hair having a predetermined shape and a method for producing artificial hair.
  • the present invention also relates to artificial hair fibers and artificial hair.
  • Non-natural fibers have the advantage that they are stronger than human hair and can be obtained in a desired length with uniform quality and in stable and large quantities.
  • artificial protein fibers for hair such as regenerated collagen fibers, which are expected to have a smaller environmental load, have also been proposed (for example, Patent Document 2).
  • the artificial hair may be given a shape including a curved portion or a linear shape by a so-called permanent wave treatment.
  • Patent Document 2 proposes to obtain a regenerated collagen fiber capable of permanent wave treatment by chemically modifying a mercapto group and/or a disulfide bond in a collagen molecule.
  • Fibroin fibers containing modified fibroin are also expected to be applied as fibers for artificial hair.
  • the present invention provides a method capable of easily imparting a predetermined shape while using fibroin fiber and obtaining a fiber for artificial hair excellent in shape retention.
  • one aspect of the present invention relates to the following.
  • the predetermined shape is a shape that includes a curved portion, a linear portion, or both of these.
  • a method for producing a fiber for artificial hair having a predetermined shape [2] The method according to [1], wherein cysteine is introduced on the surface of the fibroin fiber by contacting the fibroin fiber with a solution containing cysteine, and then the fibroin fiber is contacted with the cold perm solution.
  • the cold perm liquid is a two-liquid type cold perm liquid consisting of a first liquid containing a reducing agent and a second liquid containing an oxidizing agent, The method according to [2], wherein the fibroin fiber having cysteine introduced on the surface is brought into contact with the second liquid, the first liquid, and the second liquid in this order.
  • the cold perm liquid is a two-liquid type cold perm liquid consisting of a first liquid containing a reducing agent and a second liquid containing an oxidizing agent,
  • the first liquid further contains cysteine
  • the method according to [1] wherein cysteine is introduced into the surface of the fibroin fiber by contacting the fibroin fiber with the first liquid, and then the fibroin fiber is contacted with the second liquid.
  • a fiber for artificial hair which is provided with a predetermined shape by the method described in [9] and which expands when brought into a wet state and contracts when dried from a wet state.
  • a fiber for artificial hair having a predetermined shape can be easily obtained while using fibroin fiber that can be stably supplied and has a small environmental load.
  • the artificial hair fiber obtained by the method according to one aspect of the present invention is also excellent in shape retention.
  • the artificial hair fiber provided by one aspect of the present invention and the artificial hair containing the same can be stably supplied while suppressing the occurrence of discomfort with human hair while using the non-natural fiber.
  • One embodiment of a method for producing a fiber for artificial hair having a predetermined shape is to introduce cysteine on the surface of fibroin fiber containing modified fibroin, and to keep the fibroin fiber in a state along the predetermined shape. Contacting the fibroin fiber with cold perm fluid.
  • the predetermined shape here can be a shape including a curved portion, a linear portion, or both of them. It can be said that the shape including the curved portion is a curled shape.
  • FIG. 1 is a schematic view showing an example of a method for producing a fiber for artificial hair to which a shape (or a curled shape) including a curved portion is provided.
  • the fibroin fiber 70 is wound around the outer peripheral surface of the cylindrical core material 75. Cysteine is introduced to the surface of the fibroin fiber 70 before the fibroin fiber 70 is wound around the core material 75 or after the fibroin fiber 70 is wound around the core material 75.
  • the fibroin fiber 70 having cysteine introduced on its surface with the cold perm solution along the curled shape the fibroin fiber 70 is given a curled shape corresponding to the shape of the outer peripheral surface of the core material 75. It The fibroin fiber 70 to which the curled shape is applied can maintain the applied shape even after being removed from the core material 75.
  • the diameter of the core material 75 is not particularly limited, but is usually in the range of 1 to 100 mm.
  • the shape of the core material is not limited to the cylindrical shape.
  • cysteine By contacting the fibroin fiber 70 with a solution containing cysteine (usually an aqueous solution of cysteine), cysteine can be introduced on the surface of the fibroin fiber 70.
  • Cysteine introduced into the surface of the fibroin fiber 70 by this method is generally considered to be adsorbed on the surface of the fibroin fiber 70 without forming a covalent bond with the fibroin fiber 70.
  • the fibroin fiber 70 is a solution containing cysteine by immersing the fibroin fiber 70 in a solution containing cysteine, or by impregnating a fiber bundle containing a plurality of single filaments of the fibroin fiber 70 with a solution containing cysteine. Can be contacted with.
  • the temperature of the solution containing cysteine in this case is not particularly limited, but may be, for example, 10° C. or higher, 20° C. or higher, 30° C. or higher, or 40° C. or higher, less than 100° C., 90° C. or lower, or It may be 80°C or lower.
  • the time of contact between the fibroin fiber 70 and the solution containing cysteine is not particularly limited, but may be, for example, 1 minute or more and 24 hours or less, 12 hours or less, or 6 hours or less, and 5 minutes or more and 24 hours. It may be within, within 12 hours, or within 6 hours, and may be 10 minutes or more and within 24 hours, within 12 hours, or within 6 hours.
  • the fibroin fiber 70 after contact with the solution containing cysteine may be washed with water.
  • the concentration of cysteine in the solution containing cysteine may be, for example, 0.1% by mass or more, or 0.5% by mass or more, or 30% by mass or less, based on the mass of the entire solution.
  • concentration of cysteine may be within these numerical ranges.
  • the cold perm solution can be the one normally used for permanent wave of hair.
  • a two-liquid type cold perm liquid consisting of a first liquid containing a reducing agent and a second liquid containing an oxidizing agent. It is considered that the cysteines bond with each other to form cystine upon contact with the second liquid containing the oxidizing agent.
  • the first liquid and the second liquid may be contacted with the fibroin fiber 70 having cysteine introduced on the surface once or more.
  • the fibroin fibers 70 are usually contacted alternately with the first liquid and the second liquid, but usually they are finally contacted with the second liquid.
  • the fibroin fiber 70 having cysteine introduced on its surface may be contacted with the second liquid, the first liquid, and the second liquid in this order.
  • the fibroin fiber 70 for example, by dipping the fibroin fiber 70 in the first liquid or the second liquid, or by impregnating a fiber bundle containing a plurality of single filaments of the fibroin fiber 70 with the first liquid or the second liquid, It can be brought into contact with the first liquid or the second liquid.
  • the first liquid and/or the second liquid in contact with the fibroin fiber 70 may be heated.
  • the temperature of the first liquid and the second liquid in contact with the fibroin fiber 70 is not particularly limited, but may be, for example, 10°C or higher and lower than 100°C, 90°C or lower, or 80°C or lower, and 20°C or higher and 100°C.
  • the second liquid may be heated to 30°C or higher, or 40°C or higher.
  • the time of contact between the fibroin fiber 70 and the first liquid or the second liquid is not particularly limited, but may be, for example, 1 minute or more and 24 hours or less, 12 hours or less, or 6 hours or less, and 5 minutes or more. It may be 24 hours or less, 12 hours or less, or 6 hours or less, and may be 10 minutes or more and 24 hours or less, 12 hours or less, or 6 hours or less.
  • the first liquid of the cold perm liquid may be a mixed liquid containing a reducing agent and cysteine.
  • cysteine can be introduced into the surface of the fibroin fiber 70 by contacting the fibroin fiber 70 with the first liquid. After the contact between the fibroin fiber 70 and the first liquid containing cysteine, the fibroin fiber 70 is brought into contact with the second liquid.
  • the temperatures of the first liquid and the second liquid and the contact time can be the same as above. If the contact time between the fibroin fiber 70 and the first liquid containing cysteine is longer than the contact time between the fibroin fiber 70 and the second liquid, the shape imparted to the fibroin fiber 70 tends to be retained more easily. ..
  • the fibroin fiber 70 after contact with the final permanent liquid may be washed with water.
  • the fibroin fiber 70 after washing with water may be dried by any method such as heating.
  • the fibroin fiber 70 provided with a predetermined shape can be used as a fiber for artificial hair that constitutes artificial hair.
  • the artificial hair fiber made of fibroin fiber can be stably supplied and can suppress the occurrence of discomfort with human hair.
  • the artificial hair can be used for, for example, hair for wigs, hair for increasing hair directly attached to the head, hair for extension, and the like.
  • the fibroin fiber 70 can be a fiber containing modified fibroin as a main component and formed by spinning modified fibroin.
  • the fibroin fiber 70 wound around the core material 75 may be a fiber that is not in direct contact with liquid water after spinning.
  • the fibroin fiber 70 may be a single yarn or a fiber bundle composed of a plurality of single yarns.
  • the diameter (single yarn diameter) of the fibroin fiber 70 may be, for example, 10 to 100 ⁇ m. From the viewpoint of easily imparting the shape and maintaining the imparted shape, the single fiber diameter of the fibroin fiber 70 may exceed 30 ⁇ m, and may be 35 ⁇ m or more, or 40 ⁇ m or more. From the same viewpoint, the single yarn diameter of the fibroin fiber 70 may be less than 80 ⁇ m or 70 ⁇ m or less.
  • modified fibroin as used herein means artificially produced fibroin (artificial fibroin).
  • the modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally-occurring fibroin or may be the same fibroin as the naturally-occurring amino acid sequence of fibroin.
  • the modified fibroin is a protein containing a domain sequence represented by formula 1: [(A) n motif-REP] m or formula 2: [(A) n motif-REP] m -(A) n motif. Good.
  • an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either 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 typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.
  • modified fibroin may be one that uses the amino acid sequence of naturally-derived fibroin as it is, or one that has modified its amino acid sequence depending on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived fibroin).
  • the amino acid sequence may be modified by modifying the gene sequence of fibroin), or artificially designed and synthesized without depending on naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence It may have a desired amino acid sequence by chemical synthesis).
  • domain sequence refers to a crystalline region (typically corresponding to the (A) n motif of an amino acid sequence) and an amorphous region (typically REP of an amino acid sequence) peculiar to fibroin.
  • the (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
  • the number of amino acid residues in the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. ..
  • the ratio of the number of alanine residues to the total number of amino acid residues in the (A) n motif may be 40% or more, 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%.
  • At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • n represents an integer of 2 to 300, and may be an integer of 10 to 300.
  • the plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and/or addition of one or more amino acid residues with respect to the gene sequence of the cloned naturally-derived fibroin. It can be obtained by modifying. Amino acid residue substitutions, deletions, insertions and/or additions can be made by methods well known to those skilled in the art, such as partial directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983) and the like.
  • the naturally-occurring fibroin is a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m -(A) n motif.
  • fibroin produced by insects or arachnids can be mentioned.
  • fibroin produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yam tai rya pynayi, ori peri erygium, Anteraea periyna, Pomegranate (Anteraea periyi), Anteraea periyna, Pomegranate (Anteraea peryny) ), silkworm proteins produced by silkworms such as Anthera apse assap, such as silkworm silkworm (Antheraea assama), such as silkworm silkworms (Samia cynthia), chestnut (Caligura japonica), chusser silkworms (Antheraea mylitta), and mug silkworms (Antheraea assama). Hornet silk protein is mentioned.
  • fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), and AAA278840.1 (amino acid sequence)).
  • fibroin produced by the spiders examples include spiders belonging to the genus Araneus (genus Araneus), such as the spider Spider, spider, spider, spider, and spider, spiders belonging to the genus Araneus, spiders such as the spider Nea sp.
  • Genus Araneus such as the spider Spider, spider, spider, spider, and spider
  • spiders belonging to the genus Araneus spiders such as the spider Nea sp.
  • Spiders belonging to the genus Proton spiders belonging to the genus Pronus, spiders belonging to the genus Cyrtarachne, such as spider spider, genus Cyrtarachne, and spider spiders, such as the spider spider Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Ordgarius (genus Ordgarius), such as the spider spider, Mamaytaiseki spider and Mutsugai spider, belonging to the genus Argiopsis, such as Argiogiope, Argiope brue and Argiope brue Spiders belonging to the genus Arachnura, Spiders belonging to the genus Acusilas such as Spider Spider, spiders belonging to the genus Cytophora (Spider Spider), such as Spiders, Spiders, Black-faced Spiders and genus Cytophora (genus Cytophora).
  • spider spider Spiders belonging to the genus Proton spiders belonging to the genus Pron
  • Spider silk protein produced by spiders belonging to the genus such as spiders, lanterns, spiders belonging to the category
  • spider silk proteins produced by spiders belonging to the genus Chorizopes such as Yamato, and spider silk spiders.
  • Spiders belonging to the genus Tetragnatha such as the herring-tailed spider Spider, Harabiro-shida-daga spider and Uro-core spider, etc.
  • Spiders belonging to the genus Nephila spiders belonging to the genus Menosira, such as black spiders, spiders belonging to the genus Dyschirioognatha, such as the dwarf spider, such as the black-breasted spider, black widow spider, and the black-breasted spider.
  • spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
  • 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) [from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [from Nephila clavipes50] (session number 4 AA number) ), U37520 (base sequence)), major ampullate spidroin 1 [from Latrodetectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein vacenca Nile vulpein 2 [drugline silk caffeine 2] [spreadroin spillane] [2].
  • fibroin-3 derived from Araneus di
  • Naturally-derived fibroin include fibroin whose sequence information is registered in NCBI GenBank.
  • sequences containing INV as DIVISION among sequence information registered in NCBI GenBank spidroin, complete, fibroin, “silk and polypeptide”, or “silk and protein” are described as keywords in DEFINITION. It can be confirmed by extracting the sequence, the character string of the specific product from the CDS, and the sequence in which the specific character string is described from SOURCE to TISSUE TYPE.
  • the modified fibroin according to the present embodiment may be modified silk (silk) fibroin (modified amino acid sequence of silk protein produced by silkworm), and modified spider silk fibroin (of spider silk protein produced by spiders). Modified amino acid sequence).
  • the modified fibroin may be a modified spider silk fibroin.
  • modified fibroin examples include a modified fibroin (first modified fibroin) derived from a large vesicular guideline protein produced in the large ampullary gland of a spider, a domain sequence with a reduced content of glycine residues.
  • first modified fibroin derived from a large vesicular guideline protein produced in the large ampullary gland of a spider, a domain sequence with a reduced content of glycine residues.
  • Modified fibroin (second modified fibroin), (A) n modified fibroin having a domain sequence with reduced content of the motif (third modified fibroin), glycine residue content, and (A) n Modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence containing a region with a large hydrophobic index locally (fifth modified fibroin), and glutamine residue content Modified fibroin having a reduced domain sequence (sixth modified fibroin).
  • Examples of the first modified fibroin include a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the number of amino acid residues of (A) n motif is an integer of 3 to 20, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. It may be.
  • the number of amino acid residues constituting REP in formula 1 may be 10 to 200 residues, 10 to 150 residues, 20 to 100 residues, 20 to 75 residues ..
  • the first modified fibroin has a total number of glycine residues, serine residues, and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) n motif-REP] m.
  • the total number may be 40% or more, 60% or more, or 70% or more.
  • the first modified fibroin comprises a unit of the amino acid sequence represented by the formula 1: [(A) n motif-REP] m , and has the C-terminal sequence represented by any one of SEQ ID NOs: 1 to 3, or It may be a polypeptide which is 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 the amino acids of the C-terminal 50 residues 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: 1 is the same as the amino acid sequence with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID NO: 3 has 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
  • modified fibroin As a more specific example of the first modified fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 and 90 Mention may be made of modified fibroin, which comprises an amino acid sequence having a sequence identity of greater than or equal to %. Sequence identity may be 95% or greater.
  • the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of ADF3 in which the start codon, the His10 tag and the amino acid sequence (SEQ ID NO: 5) consisting of the HRV3C protease (Human rhinovirus 3C protease) recognition site are added to the N-terminal of the first to The 13th repeat region was increased to approximately double and the translation was mutated so that it terminated at the 1154th amino acid residue.
  • the C-terminal amino acid sequence of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.
  • the modified fibroin of (1-i) may consist of the amino acid sequence shown by SEQ ID NO:4.
  • the second modified fibroin has an amino acid sequence in which its domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP being replaced with another amino acid residue, as compared with naturally occurring fibroin. ..
  • the second modified fibroin has a domain sequence that is different from that of naturally-occurring fibroin in that GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine).
  • G is a glycine residue
  • P is a proline residue
  • X is an amino acid residue other than glycine.
  • At least one motif sequence selected from the group consisting of (1) and (2) has an amino acid sequence corresponding to the substitution of one glycine residue in at least one or more of the motif sequences with another amino acid residue. May be.
  • the proportion of the motif sequence in which the glycine residue described above is replaced with another amino acid residue may be 10% or more based on the entire motif sequence.
  • the second modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and the (A) n motif located closest to the C-terminal side from the above domain sequence is the above domain sequence.
  • z is the total number of amino acid residues of 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 Therefore, when the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located closest to the C terminus to the C terminus of the above domain sequence is w, z/w is 30% or more, It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more.
  • the number of alanine residues with respect to the total number of amino acid residues in the n motif may be 83% or more, 86% or more, 90% or more, 95% or more, or 100%.
  • the fact that the number of alanine residues is 100% of the total number of amino acid residues means that the (A) n motif is composed of only alanine residues.
  • the second modified fibroin may be one in which the content ratio 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 may be 30% or less, 20% or less, 10% or less, 6% or less, 4% or less, or 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 of calculating the content ratio (z/w) of the amino acid sequence consisting of XGX below.
  • fibroin modified fibroin or naturally occurring fibroin
  • domain sequence represented by the formula 1 [(A) n motif-REP] m , (A) n located closest to the C-terminal side from the domain sequence.
  • An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence.
  • z/w may be 50.9% or more, 56.1% or more, 58.7% or more, 70% or more, or 80% or more.
  • the upper limit of z/w is not particularly limited, but may be 95% or less, for example.
  • the second modified fibroin is obtained, for example, by modifying at least a part of the nucleotide sequence encoding a glycine residue from the cloned gene sequence of naturally-occurring fibroin so as to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or the glycine residue may be substituted so that z/w is 50.9% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence satisfying the above-mentioned aspect from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence corresponding to the insertion and/or addition may be modified.
  • the above-mentioned other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but is a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a methionine ( M) residue, proline (P) residue, hydrophobic amino acid residue such as phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S) ) Residue, a lysine (K) residue, and a glutamic acid (E) residue, and other hydrophilic amino acid residues.
  • Another amino acid residue may be selected from valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine (F) residue and glutamine (Q) residue, and glutamine ( Q) may be a residue.
  • SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met) -PRT799) or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.
  • the modified fibroin of (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin with GQX.
  • the amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 6 in which every two (A) n motifs are deleted from the N-terminal side toward the C-terminal side, and the amino acid sequence before the C-terminal sequence is further deleted.
  • One [(A) n motif-REP] was inserted into.
  • 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 is a substitution of serine (S) residue and a part of amino acids on the C-terminal side was deleted so that the molecular weight was almost the same as that of SEQ ID NO:7.
  • the amino acid sequence represented by SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 7 (however, some amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a predetermined hinge sequence and His tag sequence are added to the C-terminal of the sequence repeated 4 times.
  • the z/w value in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-derived fibroin) is 46.8%.
  • 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 each have a z/w value of 58.7%, 70.1%, 66.1% and 70.0%.
  • the values of x/y in the serrated ratios (described later) 1:1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are: They are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
  • the modified fibroin of (2-i) may consist of the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified 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 fibroin of (2-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the sequence identity may be 95% or higher.
  • the modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is contained in REP (XGX( Where X is an amino acid residue other than glycine), and z is the total number of amino acid residues of the amino acid sequence consisting of) and w is the total number of amino acid residues of REP in the above-mentioned domain sequence. May be 50.9% or more.
  • the second modified fibroin may include a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection and visualization of the modified fibroin.
  • an affinity tag that utilizes specific affinity (binding, affinity) with another molecule can be mentioned.
  • a specific example of the affinity tag is a histidine tag (His tag).
  • His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and it has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography is performed. Can be used for.
  • Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • an "epitope tag” that utilizes the antigen-antibody reaction.
  • a peptide (epitope) showing antigenicity as a tag sequence
  • an antibody against the epitope can be bound.
  • the epitope tag include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag and the like.
  • tag sequence that can be cleaved with a specific protease.
  • a protease By treating the protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence is cleaved can be recovered.
  • modified fibroin containing a tag sequence (2-iii) the amino acid shown in SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799).
  • Sequences or (2-iv) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 can be mentioned. ..
  • amino acid sequences shown in SEQ ID NO: 16 are SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • amino acid sequence shown in SEQ ID NO: 11 is added to the N-terminal of the amino acid sequence shown.
  • the modified fibroin of (2-iii) may consist of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the sequence identity may be 95% or higher.
  • the modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is contained in REP (XGX( Where X is an amino acid residue other than glycine), and z is the total number of amino acid residues of the amino acid sequence consisting of) and w is the total number of amino acid residues of REP in the above-mentioned domain sequence. May be 50.9% or more.
  • the second modified fibroin may contain a secretory 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 depending on the type of host.
  • the third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of the (A) n motif as compared to naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) n motifs as compared with the naturally occurring fibroin.
  • the third modified fibroin may have an amino acid sequence corresponding to the (A) n motif deleted from naturally occurring fibroin by 10 to 40%.
  • the third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal side toward the C-terminal one to three (A) n motif every one (A) n motif May have an amino acid sequence corresponding to the deletion.
  • the third modified fibroin has at least two consecutive (A) n motif deletions from the N-terminal side toward the C-terminal side in its domain sequence, and one (A) ) It may have an amino acid sequence corresponding to the deletion of the n motif repeated in this order.
  • the third modified fibroin may have a domain sequence having an amino acid sequence corresponding to at least every two (A) n motifs deleted from the N-terminal side toward the C-terminal side. ..
  • the third modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and has two adjacent [(A) n motifs from the N-terminal side to the C-terminal side.
  • -REP] units are sequentially compared with each other, and when the number of amino acid residues of a REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues of the other REP is 1.8 to When the maximum value of the sum of the amino acid residues of two adjacent [(A) n motif-REP] units that are 11.3 is x and the total number of amino acid residues of the domain sequence is y In addition, x/y may have an amino acid sequence having 20% or more, 30% or more, 40% or more, or 50% or more.
  • the number of alanine residues with respect to the total number of amino acid residues in the n motif may be 83% or more, 86% or more, 90% or more, 95% or more, or 100%.
  • the fact that the number of alanine residues is 100% of the total number of amino acid residues means that the (A) n motif is composed of only alanine residues.
  • FIG. 2 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from modified fibroin. From the N-terminal side (left side), the domain sequence is (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has an n motif sequence.
  • Two adjacent [(A) n motif-REP] units are selected sequentially from the N-terminal side toward the C-terminal side so that there is no overlap. At this time, an unselected [(A) n motif-REP] unit may be present.
  • pattern 1 (comparison of first REP and second REP, and comparison of third REP and fourth REP)
  • pattern 2 comparison of first REP and second REP
  • Pattern 4 (comparison of fourth REP and fifth REP)
  • pattern 3 comparison of second REP and third REP, and comparison of fourth REP and fifth REP
  • pattern 4 compared with first REP
  • the number of amino acid residues of each REP in two adjacent [(A) n motif-REP] units selected is compared.
  • the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues)
  • the fourth REP having a smaller number of amino acid residues is set to 1
  • each pattern the total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by solid lines is added (not only REP but also the number of amino acid residues of (A) n motif is is there.). Then, the added total values are compared, and the total value of the patterns having the maximum total value (the maximum value of the total values) is set as x. In the example shown in FIG. 2, 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 in the domain sequence, y.
  • x/y may be 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more.
  • the upper limit of x/y is not particularly limited, and for example, x/y may be 100% or less.
  • the serrated ratio may be 1:1.9 to 11.3 and x/y may be 89.6% or more.
  • the serrated ratio may be 1:1.8 to 3.4 and x/y may be 77.1% or more.
  • the serrated ratio may be 1:1.9 to 8.4 and x/y may be 75.9% or more.
  • the serrated ratio may be 1:1.9 to 4.1 and x/y may be 64.2% or more.
  • x/y is 46.4% or more, 50% or more. It may be 55% or more, 60% or more, 70% or more, or 80% or more.
  • the upper limit of x/y is not particularly limited and may be 100% or less.
  • x/y in naturally occurring fibroin will be described.
  • 663 kinds of fibroins (of which 415 kinds of spider-derived fibroins) were extracted.
  • x/y was calculated from the amino acid sequence of naturally-derived fibroin composed of the domain sequence represented by the formula 1: [(A) n motif-REP] m by the above-mentioned calculation method.
  • x/y in naturally-derived fibroin was less than 64.2%, and the maximum was 64.14%.
  • the third modified fibroin has, for example, one or more sequences encoding the (A) n motif deleted from the cloned gene sequence of naturally-derived fibroin so that x/y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed and designed from the amino acid sequence of naturally-occurring fibroin so that x/y is 64.2% or more. It can also be obtained by chemically synthesizing a nucleic acid encoding the above amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and/or added.
  • the amino acid sequence corresponding to the above may be modified.
  • the third modified fibroin (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met) -PRT799) or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.
  • the modified fibroin of (3-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 17 is the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin, and every two (A) n from the N-terminal side to the C-terminal side.
  • the motif is deleted and one [(A) n motif-REP] is inserted before the C-terminal sequence.
  • the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 are as described for the second modified fibroin.
  • the x/y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-derived fibroin) at the Giza ratio of 1:1.8 to 11.3 is 15.0%.
  • the values of x/y in the amino acid sequence shown by SEQ ID NO:17 and the amino acid sequence shown by SEQ ID NO:7 are both 93.4%.
  • the value of x/y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%.
  • the x/y value in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%.
  • the z/w values in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
  • the modified fibroin of (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified 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: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (3-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the sequence identity may be 95% or higher.
  • the modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from the N-terminal side to the C-terminal side.
  • X/y may be 64.2% or more, where x is the maximum value of the total sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may include the above-mentioned tag sequence at either or both of the N-terminus and the C-terminus.
  • modified fibroin containing a tag sequence (3-iii) the amino acid shown in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799).
  • Sequences, or (3-iv) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 can be mentioned. ..
  • amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 have SEQ ID NO: 11 at the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • the amino acid sequence represented by (including His tag sequence and hinge sequence) is added.
  • the modified fibroin of (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified 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: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the sequence identity may be 95% or higher.
  • the modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and has N-terminal side to C-terminal side.
  • X/y may be 64.2% or more, where y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may contain a secretory 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 depending on the type of host.
  • the fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) n motif and a reduced content of glycine residues as compared with naturally-occurring fibroin. I have.
  • the domain sequence of the fourth modified fibroin has at least one or more (A) n motifs deleted in addition to at least one or more glycine residues in REP, as compared to naturally-occurring fibroin. It can be said that it has an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the above-described second modified fibroin and third modified fibroin. Specific aspects and the like are as described for the second modified fibroin and the third modified fibroin.
  • fourth modified fibroin examples include (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410).
  • SEQ ID NO: 14 PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15
  • a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by Specific embodiments of the modified fibroin containing the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.
  • the fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a large hydrophobicity index, and/or REP as compared with naturally-occurring fibroin. It may have an amino acid sequence locally containing a region with a large hydrophobicity index, which corresponds to the insertion of one or more amino acid residues with a large hydrophobicity index.
  • a region having a locally high hydrophobicity index may be composed of consecutive 2 to 4 amino acid residues.
  • the above-mentioned amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). It may be a residue.
  • the fifth modified fibroin has one or more amino acid residues in REP replaced with an amino acid residue having a large hydrophobicity index, and/or one or more amino acids in REP, as compared to naturally-occurring fibroin.
  • substitution, deletion, insertion and/or addition of one or more amino acid residues as compared with naturally occurring fibroin There may be an amino acid sequence modification corresponding to what was done.
  • the fifth modified fibroin has, for example, one or more hydrophilic amino acid residues (for example, an amino acid residue having a negative hydrophobicity index) in REP from the gene sequence of the cloned naturally-derived fibroin, and a hydrophobic amino acid residue. It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid residues in REP. Further, for example, one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acid residues in REP.
  • one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acid residues in REP.
  • an amino acid sequence corresponding to the insertion of the amino acid sequence can also be obtained by designing an amino acid sequence corresponding to the insertion of the amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally-derived fibroin, and/or one or more hydrophobic amino acids in REP.
  • the modification of the amino acid sequence corresponding to the replacement, deletion, insertion and/or addition of one or more amino acid residues may be performed.
  • the fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , from the (A) n motif located closest to the C terminus to the C terminus of the above domain sequence.
  • the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is p
  • the p/q is 6 It may have an amino acid sequence of 2% or more.
  • hydrophobicity index of amino acid residues
  • HI hydrophobicity index
  • a sequence obtained by removing the sequence from the domain sequence represented by the formula 1: [(A) n motif-REP] m to the C-terminal of the (A) n motif located at the most C-terminal side (Hereinafter referred to as “array A”) is used.
  • array A a sequence obtained by removing the sequence from the domain sequence represented by the formula 1: [(A) n motif-REP] m to the C-terminal of the (A) n motif located at the most C-terminal side.
  • the average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times).
  • a region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to multiple “4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more”, it must be included as 1 amino acid residue in the region. become.
  • 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 may be 6.2% or more, 7% or more, 10% or more, 20% or more, or 30% or more.
  • the upper limit of p/q is not particularly limited, but may be 45% or less, for example.
  • the fifth modified fibroin includes, for example, one or more hydrophilic amino acid residues (for example, hydrophobicity index) in REP so that the amino acid sequence of the cloned naturally-occurring fibroin is satisfied so that the above p/q condition is satisfied. Is replaced with a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index), and/or one or more hydrophobic amino acid residues are inserted into REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index.
  • hydrophilic amino acid residues for example, hydrophobicity index
  • one or more amino acid residues in REP were replaced with amino acid residues having a large hydrophobicity index, and/or one or more amino acid residues in REP, as compared with naturally-occurring fibroin.
  • modification corresponding to substitution, deletion, insertion and/or addition of one or more amino acid residues may be carried out. ..
  • 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 ), or an amino acid residue selected from valine (V), leucine (L) and isoleucine (I).
  • the fifth modified fibroin (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), Alternatively, (5-ii) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can be mentioned.
  • the amino acid sequence represented by SEQ ID NO: 19 is composed of 3 amino acid residues in every other REP with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410), except for the terminal domain sequence on the C-terminal side.
  • An amino acid sequence (VLI) was inserted at two positions, a part of glutamine (Q) residue was replaced with a serine (S) residue, and a part of the C-terminal side amino acid was deleted.
  • the amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with an amino acid sequence (VLI) consisting of 3 amino acid residues at every other REP inserted at one position. is there.
  • the amino acid sequence represented by SEQ ID NO: 21 is the amino acid sequence represented by SEQ ID NO: 8 with two amino acid sequences (VLI) each consisting of three amino acid residues inserted every other REP.
  • the modified fibroin of (5-i) may consist of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the sequence identity may be 95% or higher.
  • the modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located at the most C-terminal side (A) n.
  • P/q may be 6.2% or more.
  • the fifth modified fibroin may include a tag sequence at either or both of the N-terminus and the C-terminus.
  • modified fibroin containing a tag sequence (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv )
  • a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 can be mentioned.
  • amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including sequences and hinge sequences).
  • the modified fibroin of (5-iii) may consist of the amino acid sequence shown by SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.
  • the modified fibroin (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the sequence identity may be 95% or higher.
  • the modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located at the most C-terminal side (A) n.
  • P/q may be 6.2% or more.
  • the fifth modified fibroin may contain a secretory 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 depending on the type of host.
  • the sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared to naturally-occurring fibroin.
  • the sixth modified fibroin may include at least one motif selected from the GGX motif and the GPGXXX motif in the amino acid sequence of REP.
  • the GPGXX motif content may be usually 1% or more, 5% or more, or 10% or more.
  • the upper limit of the content rate of the GPGXX motif is not particularly limited and may be 50% or less, or 30% or less.
  • the “GPGXX motif content rate” is a value calculated by the following method.
  • Formula 1 [(A) n Motif-REP] m
  • Formula 2 [(A) n Motif-REP] m -(A)
  • a fibroin containing a domain sequence represented by the n- motif modified fibroin or naturally derived fibroin (A) n motif located closest to the C-terminal side in the fibroin
  • the sequence from the C sequence to the C-terminal of the domain sequence is excluded from all the REPs
  • the number of GPGXX motifs contained in the region is Let s be the number three times the total number (that is, the total number of G and P in the GPGXX motif), and the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence.
  • the total number of amino acid residues of all REPs excluding (A) n motifs is t, the GPG
  • the sequence obtained by excluding the sequence from the (A) n motif located at the most C-terminal to the C-terminal of the domain sequence from the domain sequence is "the most C-terminal side".
  • the sequence (A) from the n- motif to the C-terminal of the domain sequence may include a sequence having a low correlation with the sequence characteristic of fibroin, and m is small. In this case (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content rate, and is for eliminating this effect.
  • the “GPGXX motif” is located at the C-terminal of REP, it is treated as a “GPGXX motif” even if “XX” is “AA”.
  • FIG. 4 is a schematic diagram showing a domain sequence of modified fibroin.
  • a method of calculating the GPGXX motif content rate will be specifically described with reference to FIG. First, in the modified fibroin domain sequence (“[(A) n motif-REP] m ⁇ (A) n motif” type) shown in FIG. 4, all REPs are “most C-terminally located.
  • (A) The sequence from the n motif to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence shown as “region A” in FIG. 4).
  • the glutamine residue content of the sixth modified fibroin may be 9% or less, 7% or less, 4% or less, or 0%.
  • the "glutamine residue content rate” is a value calculated by the following method.
  • Formula 1 [(A) n Motif-REP] m
  • Formula 2 [(A) n Motif-REP] m -(A) A fibroin containing a domain sequence represented by the n- motif (modified fibroin or naturally derived fibroin In fibroin), all contained in the sequence (sequence corresponding to “region A” in FIG. 4) in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminus of the domain sequence is excluded from the domain sequence.
  • the total number of glutamine residues contained in the region is defined as u
  • the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence
  • (A) n The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues of all REPs excluding the motif.
  • the reason why “the sequence obtained by 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 the same as the above-mentioned reason. The same is true.
  • the sixth modified fibroin corresponds to a domain sequence in which one or more glutamine residues in REP are deleted or substituted with other amino acid residues, as compared with naturally-occurring fibroin. It may have an amino acid sequence.
  • the other amino acid residue may be an amino acid residue other than the glutamine residue, but may be an amino acid residue having a larger hydrophobicity index than the glutamine residue.
  • the hydrophobicity index of amino acid residues is shown in Table 1.
  • An amino acid residue selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H) can be mentioned. it can.
  • an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) may be used, It may be an amino acid residue selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
  • the hydrophobicity of the REP of the sixth modified fibroin may be ⁇ 0.8 or higher, ⁇ 0.7 or higher, 0 or higher, 0.3 or higher, or 0.4 or higher.
  • the upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.
  • hydrophilcity of REP is a value calculated by the following method.
  • Formula 1 [(A) n Motif-REP] m
  • Formula 2 [(A) n Motif-REP] m -(A) A fibroin containing a domain sequence represented by the n- motif (modified fibroin or naturally derived fibroin In fibroin), all contained in the sequence (sequence corresponding to “region A” in FIG. 4) in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminus of the domain sequence is excluded from the domain sequence.
  • the sum of the hydrophobicity indices of each amino acid residue in the region is defined as v
  • the sequence from the (A) n motif located closest to the C terminus to the C terminus of the domain sequence is removed from the domain sequence
  • A) The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues of all REPs excluding n motifs.
  • the reason why "the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the target is the same as the above-mentioned reason. The same is true.
  • the sixth modified fibroin has a domain sequence in which one or more glutamine residues in REP are deleted, and/or one or more glutamine residues in REP, as compared to naturally-occurring fibroin.
  • the modification corresponding to the substitution of the amino acid residue with another amino acid residue there may be a modification of the amino acid sequence corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues. ..
  • the sixth modified fibroin includes, for example, deleting one or more glutamine residues in REP from a cloned gene sequence of naturally-occurring fibroin, and/or deleting one or more glutamine residues in REP from each other. It can be obtained by substituting the amino acid residue of Further, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally-derived fibroin, and/or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met) -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or sequence No.
  • the amino acid sequence represented by SEQ ID NO:25 is the amino acid sequence represented by SEQ ID NO:7 (Met-PRT410) in which all QQs are replaced with VLs.
  • the amino acid sequence represented by SEQ ID NO:26 is obtained by replacing QQ in the amino acid sequence represented by SEQ ID NO:7 with TS and replacing the remaining Q with A.
  • the amino acid sequence represented by SEQ ID NO:27 is obtained by replacing QQ in the amino acid sequence represented by SEQ ID NO:7 with VL and replacing the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO:28 is obtained by substituting VI for all QQ in the amino acid sequence represented by SEQ ID NO:7 and substituting L for the remaining Q.
  • the amino acid sequence represented by SEQ ID NO: 29 is obtained by replacing QQ in the amino acid sequence represented by SEQ ID NO: 7 with VF and replacing the remaining Q with I.
  • the amino acid sequence shown in SEQ ID NO: 30 is the amino acid sequence shown in SEQ ID NO: 8 (Met-PRT525) with all QQ replaced with VL.
  • the amino acid sequence represented by SEQ ID NO: 31 is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 8 with VL and replacing the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 32 is a sequence obtained by repeating twice the region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410), and substituting VF for VF. In addition, the remaining Q is replaced with I.
  • the amino acid sequence represented by SEQ ID NO: 41 (Met-PRT917) is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 7 with LI and replacing the remaining Q with V.
  • the amino acid sequence represented by SEQ ID NO: 42 (Met-PRT1028) is the amino acid sequence represented by SEQ ID NO: 7 in which all QQ are replaced with IF and the remaining Q is replaced with T.
  • amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all glutamine residues.
  • the group content is 9% or less (Table 2).
  • the modified fibroin of (6-i) is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42. It may consist of the amino acid sequence shown.
  • the modified fibroin of (6-ii) is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42. It includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown.
  • the modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif.
  • the modified fibroin of (6-ii) may have a glutamine residue content of 9% or less.
  • the modified fibroin of (6-ii) may have a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection and visualization of the modified fibroin.
  • modified fibroin containing a tag sequence examples include (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37.
  • amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are respectively SEQ ID NO: 25. , SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:41 and SEQ ID NO:42 at the N-terminal of the amino acid sequence shown by SEQ ID NO:11. Amino acid sequence (including His tag sequence and hinge sequence).
  • SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).
  • the modified fibroin of (6-iii) is SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43 or SEQ ID NO:44. It may consist of the amino acid sequence shown.
  • the modified fibroin of (6-iv) is SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43 or SEQ ID NO:44. It includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown.
  • the modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif.
  • the modified fibroin of (6-iv) may have a glutamine residue content of 9% or less.
  • the modified fibroin (6-iv) may have a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a secretory 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 depending on the type of host.
  • the modified fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. It may be a modified fibroin having the characteristics of
  • the modified fibroin may be hydrophobic modified fibroin because it can suppress contraction due to contact with water.
  • “hydrophobic modified fibroin” means a value obtained by summing the hydrophobicity indices (HI) of all amino acid residues constituting the modified fibroin, and then dividing the sum by the total number of amino acid residues. It is a modified fibroin having (average HI) of 0 or more.
  • the average value (average HI) of the hydrophobic index of the modified fibroin may be 0.5 or more, or 0.6 or more.
  • the average value of the hydrophobicity index (average HI) of the modified fibroin may be, for example, 1.2 or less, or 0.9 or less.
  • the hydrophobicity index is as shown in Table 1. Further, modified fibroin having an average HI of less than 0 may be referred to as hydrophilic modified fibroin.
  • hydrophobic modified fibroin for example, the amino acid sequence shown in 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, SEQ ID NO: 35, Examples include modified fibroin containing the amino acid sequence represented by 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 fibroin examples include the amino acid sequence represented by SEQ ID NO: 4, the 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, SEQ ID NO: 14.
  • amino acid sequence represented by SEQ ID NO: 15 the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, the amino acid represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin containing the amino acid sequence shown by the sequence, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can be mentioned.
  • the modified fibroin for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.
  • the method for producing the nucleic acid encoding the modified fibroin is not particularly limited.
  • the nucleic acid is produced by utilizing a gene encoding natural fibroin, amplifying it by polymerase chain reaction (PCR) or the like, cloning it, and modifying it by a genetic engineering method, or chemically synthesizing it. can do.
  • PCR polymerase chain reaction
  • the method of chemically synthesizing nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of fibroin obtained from the NCBI web database, etc., in AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) Genes can be chemically synthesized by a method of ligating automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and/or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon and a His10 tag to the N-terminal of the above amino acid sequence was synthesized. You may.
  • the regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) and can be appropriately selected according to the type of host.
  • a promoter an inducible promoter that functions in a host cell and is capable of inducing expression of modified fibroin may be used.
  • 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 physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.
  • the type of expression vector can be appropriately selected according to the type of host, such as plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector.
  • the expression vector may be capable of autonomous replication in a host cell, or can be integrated into the host chromosome, and may contain a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed.
  • prokaryote and eukaryote such as yeast, filamentous fungus, insect cell, animal cell and plant cell can be used.
  • prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganism belonging to the genus Brevibacillus include Brevibacillus agri.
  • microorganisms belonging to the genus Serratia include Serratia liquefaciens.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphilum and the like.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes and the like.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.
  • examples of a vector for introducing a nucleic acid encoding modified fibroin include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238569) and the like can be mentioned.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeasts include yeasts belonging to the genera Saccharomyces, Pichia, Schizosaccharomyces, and the like.
  • Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.
  • examples of the vector into which the nucleic acid encoding the modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051).
  • 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 method electroporation method
  • spheroplast method protoplast method
  • lithium acetate method competent method 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, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. ..
  • the modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting from the culture medium.
  • the method for culturing the host in the culture medium can be performed according to the method usually used for culturing the host.
  • the culture medium contains a carbon source, a nitrogen source and inorganic salts that can be assimilated by the host and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.
  • the carbon source may be any as long as it can be assimilated by the above-mentioned transformed microorganism, and examples thereof include glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysates, and acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic acids 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 and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof can be used.
  • the inorganic salts for example, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.
  • Culturing of prokaryotic organisms such as Escherichia coli or eukaryotic organisms such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration agitation culture.
  • the culture temperature is, for example, 15 to 40°C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during culturing may be maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • indole acrylic is used. You may add an acid etc. to a culture medium.
  • Isolation and purification of the expressed modified fibroin can be performed by a commonly used method.
  • the modified fibroin is expressed in a lysed state in cells
  • the host cells are recovered by centrifugation, suspended in an aqueous buffer solution, and then ultrasonically disrupted, French press, Menton.
  • a cell-free extract is obtained by crushing host cells with a Gaurin homogenizer, Dynomill and the like. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolation and purification of proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent is used.
  • Precipitation method anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), and cation using a resin such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing
  • a resin such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.)
  • S-Sepharose FF manufactured by Pharmacia
  • Ion exchange chromatography hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose
  • the modified fibroin When the modified fibroin is expressed by forming an insoluble substance in the cell, similarly recover the host cell, recover the insoluble substance of the modified fibroin as a precipitate fraction by crushing and centrifuging.
  • the recovered insoluble body of modified fibroin can be solubilized with a protein denaturing agent.
  • a purified fibroin preparation can be obtained by the same isolation and purification method as described above.
  • the modified fibroin can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture with a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.
  • the fibroin fiber provided with a predetermined shape or a curled shape may be one that expands when it is in a wet state and contracts when it is dried from a wet state.
  • a predetermined shape By imparting a predetermined shape to the fibroin fiber that expands when it is in a wet state and shrinks when dried from a wet state, it expands when it is in a wet state and is dried from a wet state It is possible to easily manufacture the artificial hair fiber that is contracted at the time and has a predetermined shape.
  • the predetermined shape may be a shape including a curved portion, a linear portion, or both of them.
  • the fibroin fiber that expands when it is in a wet state and contracts when it is dried from the wet state may have a fibroin fiber recovery rate defined by the following formula (1) of 95% or more.
  • Recovery rate (length of fibroin fiber when dried from wet state/length of fibroin fiber before being in wet state) ⁇ 100 (%)
  • the restoration rate of the fibroin fiber defined by the formula (1) may be 96% or more, 97% or more, 98% or more, or 99% or more.
  • the elongation rate of the fibroin fiber defined by the following formula (4) may be 17% or less.
  • the elongation rate defined by the equation (4) is an index of elongation characteristics when the fibroin fiber is in a wet state.
  • Formula (4): Elongation rate ⁇ (length of fibroin fiber in wet state/length of fibroin fiber before becoming wet)-1 ⁇ 100(%)
  • the extension rate defined by the equation (4) may be, for example, 15% or less, 13% or less, 10% or less, or 5% or less.
  • the elongation rate defined by the formula (4) may be more than 0%, 1% or more, 2% or more, 5% or more, 10% or more, or 13% or more.
  • the elongation rate of the fibroin fiber according to the present embodiment may be, for example, more than 0% and 17% or less, more than 0% and 15% or less, or 2% or more and 15% or less. 5% or more and 15% or less, 5% or more and 13% or less, 5% or more and 10% or less, or more than 0% and 10% or less It may be more than 0% and 5% or less. From the viewpoint of reducing the discomfort between the artificial hair and the human hair, the elongation rate defined by the formula (4) may be small.
  • the shrinkage rate C of the fibroin fiber defined by the following formula (5) may be 17% or less.
  • the shrinkage rate C defined by the equation (5) is an index of shrinkage characteristics when the fibroin fiber is dried from the wet state.
  • Formula (5): Shrinkage C ⁇ 1-(length of fibroin fiber when dried from wet state/length of fibroin fiber in wet state) ⁇ 100 (%)
  • the shrinkage rate C defined by the equation (5) may be 15% or less, 13% or less, 10% or less, or 5% or less.
  • the shrinkage rate C defined by the formula (5) may be more than 0%, 1% or more, 2% or more, 5% or more, 10% or more, or 13% or more.
  • the shrinkage rate C of the fibroin fiber according to the present embodiment is, for example, more than 0% and 17% or less, more than 0% and 15% or less, 2% or more and 15% or less, 5% or more and 15% or less, 5% or more. And 13% or less, 5% or more and 10% or less, more than 0% and 10% or less, or more than 0% and 5% or less. From the viewpoint of reducing the discomfort between the artificial hair and the human hair, the shrinkage rate C defined by the formula (5) may be small.
  • the fibroin fiber may be a fiber having a contraction history that contracts irreversibly by contact with water after spinning.
  • the shrinkage rate A of the fibroin fiber defined by the following formula (2) may be 2% or more.
  • the fibroin fiber having a shrinkage rate A of 2% or more has a behavior closer to that of human hair when wet/dry.
  • Shrinkage A ⁇ 1-(length of fiber contracted irreversibly by contact with water after spinning/length of fiber after spinning but before contact with water) ⁇ 100( %)
  • the shrinkage rate A defined by the formula (2) is 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6 % Or more, 10% or more, 15% or more, 20% or more, or 25% or more.
  • the upper limit of the shrinkage rate A defined by the formula (2) is not particularly limited, but the shrinkage rate A is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less. It may be 5% or less, 4% or less, or 3% or less.
  • the fibroin fiber may be a fiber having a shrinkage history in which it is contracted irreversibly by contact with water after spinning and then further contracted by drying.
  • the shrinkage rate B of the fibroin fiber defined by the following formula (3) may be more than 7%.
  • the fibroin fiber having a shrinkage ratio B defined by the formula (3) of more than 7% has a behavior closer to that of human hair when wet/dry.
  • Shrinkage ratio B ⁇ 1-(length of fiber that has been irreversibly contracted by contact with water after spinning and then further contracted by drying/fiber after spinning but before contact with water Length) ⁇ 100(%)
  • the shrinkage ratio B defined by the formula (3) is 10% or more, 15% or more, more than 25%, 32% or more, 40% or more, 48% or more, 56% or more, 64% or more or 72% or more. You can
  • the upper limit of the shrinkage rate B defined by the formula (3) is not particularly limited, but is usually 80% or less.
  • a recess or groove extending in the fiber axis direction may be formed on the surface of the fibroin fiber.
  • the fibroin fiber having the surface in which the concave portion or the groove portion is formed has a suppressed gloss and can realize the appearance similar to that of human hair.
  • Examples of the method for forming the recesses on the surface of the fibroin fiber include, for example, a method of spinning by a wet spinning method when spinning a raw material fiber, a method of slowing the desolvation speed (for example, adding a solvent of a dope solution to a coagulating solution). Method), and the method described in International Publication No. 2016/2013369 (for example, a method of increasing the residence time in the coagulation bath (60 seconds or more), a method of changing the solvent ratio in the coagulation bath). it can.
  • the heat shrinkage rate of the fibroin fiber defined by the following formula (6) may be 4% or less.
  • Formula (6): heat shrinkage ratio ⁇ 1-(fibroin fiber length when heated to 160° C./fibroin fiber length before heating) ⁇ 100 (%)
  • Fibers for artificial hair which consist of fibroin fibers containing modified fibroin, have a softening point at a temperature that is higher than general synthetic fibers and comparable to human hair. Therefore, the heat shrinkage at 160° C. (heat shrinkage defined by the equation (6)) is small. It is said that the hot air temperature of the hair dryer is 120 to 140°C, and the suitable temperature for using the hair iron is 170°C or less. Since the heat shrinkage rate defined by the formula (6) is small, damage when using a hair dryer or a hair iron can be suppressed.
  • the heat shrinkage rate defined by the equation (6) may be, for example, 4% or less, 3% or less, or 2.5% or less.
  • Fibroin fiber containing modified fibroin can also be prepared as an animal-free (no animal-derived component) material.
  • the fibroin fiber 70 can be manufactured by a usual spinning method using a dope solution containing modified fibroin.
  • the dope may be, for example, modified fibroin in a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP), if necessary, with an inorganic salt as a dissolution accelerator. It is prepared by adding and dissolving with.
  • DMSO dimethyl sulfoxide
  • DMF N,N-dimethylformamide
  • HFIP hexafluoroisopropanol
  • spinning can be carried out by a spinning method such as wet spinning, dry spinning, and dry wet spinning to obtain the target fibroin fiber.
  • the spinning method may be a melt spinning method or the like.
  • FIG. 5 is a schematic diagram showing an example of a spinning device for producing fibroin fiber.
  • the spinning device 10 shown in FIG. 5 is an example of a spinning device for dry-wet spinning, and includes an extrusion device 1, an undrawn yarn producing device 2, a wet heat drawing device 3, and a drying device 4.
  • the dope liquid 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8.
  • the dope may be filled in a cylinder and pushed out from a nozzle using a syringe pump.
  • the extruded dope liquid 6 is supplied into the coagulating liquid 11 in the coagulating liquid tank 20 through the air gap 19, the solvent is removed, and the modified fibroin is coagulated to form a fibrous coagulated body.
  • the fibrous solidified body is supplied into the warm water 12 in the stretching bath 21 and stretched.
  • the draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14.
  • the stretched fibrous coagulated body is supplied to the drying device 4 and dried in the yarn path 22 to obtain the fibroin fiber 70 as the wound body 5.
  • 18a to 18g are thread guides.
  • the coagulating liquid 11 may be any solvent that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone.
  • the coagulation liquid 11 may contain water.
  • the temperature of the coagulation liquid 11 may be 0 to 30°C.
  • the distance that the coagulated protein passes through the coagulation liquid 11 may be any length that allows efficient desolvation, for example, 200 to 500 mm. Is.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m/min, or 1 to 3 m/min.
  • the residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, or 0.05 to 0.15 minutes. Stretching (pre-stretching) may be performed in the coagulating liquid 11.
  • the coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or in a specific stage as needed.
  • the stretching of the fibroin fiber 70 for example, dry heat stretching may be used in addition to the above-described pre-stretching performed in the coagulating liquid tank 20 and wet heat stretching performed in the stretching bath 21.
  • the wet heat stretching can be performed in warm water, a solution of warm water plus an organic solvent, or steam heating.
  • the temperature may be, for example, 50 to 90°C, or 75 to 85°C.
  • the undrawn yarn (or pre-drawn yarn) may be drawn 1 to 10 times, or 2 to 8 times, for example.
  • the dry heat drawing can be performed using an electric tubular furnace, a dry heat plate, or the like.
  • the temperature of the dry heat stretching may be, for example, 140°C to 270°C, or 160°C to 230°C.
  • the undrawn yarn (or pre-drawn yarn) may be drawn, for example, 0.5 to 8 times, or 1 to 4 times.
  • the wet heat stretching and the dry heat stretching may be performed individually, or may be performed in multiple stages or in combination.
  • the first stage stretching is performed by wet heat stretching
  • the second stage stretching is performed by dry heat stretching
  • the first stage stretching is performed by wet heat stretching
  • the second stage stretching is performed by wet heat stretching
  • the third stage stretching is performed by dry heat stretching.
  • the wet heat stretching and the dry heat stretching may be appropriately combined.
  • the final draw ratio is more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times with respect to the undrawn yarn (or pre-drawn yarn). Or more, or 9 times or more, 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less, 12 times or less, 11 times or less, or 10 times or less. May be.
  • the fibroin fiber is a fiber spun at a draw ratio of 2 times or more, the shrinkage rate when the fibroin fiber is brought into contact with water to be in a wet state becomes higher.
  • the fibroin fiber 70 formed by spinning may be contracted.
  • the fibroin fiber obtained through the shrinking step may have a property of expanding when it is in a wet state and shrinking when it is dried from the wet state.
  • the shrinking step may include, for example, irreversibly shrinking the fibroin fiber 70 after spinning, before contacting with water, with water. After contact with water, the fibroin fibers 70 may be further shrunk by drying.
  • the fibroin fiber 70 before contacting with water after spinning is brought into contact with water to bring the fibroin fiber 70 into a wet state.
  • the wet state means a state in which at least a part of the fibroin fiber 70 is wet with water. Thereby, the fibroin fiber 70 can be contracted irreversibly regardless of the external force.
  • the temperature of the water contacting the fibroin fiber 70 may be below the boiling point. This improves the handleability and workability of the shrinking process. From the viewpoint of sufficiently shortening the contraction time, the temperature of water may be 10°C or higher, 40°C or higher, or 70°C or higher. The temperature of water may be 90° C. or lower.
  • the method of bringing water into contact with the fibroin fiber 70 is not particularly limited.
  • a method of immersing the fibroin fiber 70 in water a method of spraying water on the fibroin fiber 70 at room temperature or in a heated state such as steam, and high humidity in which the fibroin fiber 70 is filled with water vapor
  • a method of exposing to the environment The method of immersing the fibroin fiber 70 in water may be adopted because the contraction time can be effectively shortened and the processing equipment can be simplified.
  • the fibroin fiber 70 When the fibroin fiber 70 is brought into contact with water in a relaxed state, the fibroin fiber 70 may not only contract but also wrinkle. In order to prevent the occurrence of such shrinkage, the fibroin fiber 70 may be brought into contact with water without being relaxed, for example, by bringing the fibroin fiber 70 into contact with water while applying tension in the fiber axis direction.
  • the wet fibroin fiber 70 may be contracted by drying. Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known contact-type or non-contact-type drying equipment can be used.
  • the drying temperature may be, for example, a temperature lower than the temperature at which the modified fibroin is decomposed or the fibroin fiber 70 is thermally damaged, and may be, for example, 20 to 150° C. or 50 to 100° C. ..
  • the drying time is appropriately set according to the drying temperature and the like, and, for example, a time such that the influence of overdrying on the quality and physical properties of the fibroin fiber 70 can be eliminated as much as possible is adopted.
  • FIG. 6 is a schematic diagram showing an example of a manufacturing apparatus for manufacturing fibroin fiber.
  • the manufacturing apparatus 40 shown in FIG. 6 is an apparatus for shrinking the fibroin fiber 70 formed by spinning.
  • the manufacturing apparatus 40 includes a feed roller 42 that sends out the fibroin fibers 70, a winder 44 that winds the contracted fibroin fibers 70, a water bath 46 for bringing the fibroin fibers 70 into contact with water, and a wet fibroin fiber 70. And a dryer 48 for drying.
  • the wound material of fibroin fiber 70 is attached to the feed roller 42.
  • the fibroin fiber 70 is continuously and automatically sent out by the rotation of the electric motor or the like.
  • the winder 44 continuously and automatically winds the contracted fibroin fiber 70 by the rotation of the electric motor.
  • the delivery speed of the fibroin fiber 70 by the feed roller 42 and the winding speed of the fibroin fiber 70 by the winder 44 can be controlled independently of each other.
  • the winding speed of the winder 44 may be slower than the feeding speed of the feed roller 42.
  • the fibroin fiber 70 contracts due to contact with the water 47 in a state in which the fibroin fiber 70 is tensioned so as not to relax between the feed roller 42 and the winder 44, and thus it is possible to prevent the occurrence of curling.
  • the fibroin fiber 70 contracts irreversibly upon contact with the water 47.
  • the water bath 46 and the dryer 48 are arranged between the feed roller 42 and the winder 44 in order from the upstream side in the feeding direction of the fibroin fiber 70.
  • the manufacturing apparatus 40 has relay rollers 50 and 52 that relay the fibroin fibers 70.
  • the water bath 46 has a heater 54, and the water 47 heated by the heater 54 is stored in the water bath 46.
  • the tension roller 56 is installed while being immersed in the water 47.
  • the fibroin fiber 70 sent out from the feed roller 42 travels toward the winder 44 side while being immersed in the water 47 in the water bath 46 while being wound around the tension roller 56.
  • the dryer 48 has a pair of hot rollers 58.
  • the fibroin fiber 70 which leaves the water bath 46 and runs toward the winder 44, is wound around the pair of hot rollers 58.
  • the wet fibroin fiber 70 is heated in the dryer 48 by the pair of hot rollers 58, and thereby dried. This drying causes the fibroin fiber 70 to further shrink.
  • the dried fibroin fiber 70 is wound around the winder 44.
  • the fibroin fiber 70 can be further shrunk and the length of the fibroin fiber 70 can be unchanged.
  • FIG. 7 is a schematic diagram showing another example of a manufacturing apparatus for manufacturing fibroin fiber.
  • the manufacturing apparatus of FIG. 7 is also an apparatus for shrinking the fibroin fiber 70 formed by spinning, a processing apparatus 60 for contacting the fibroin fiber 70 with water, and a drying apparatus for drying the wet fibroin fiber 70. And a drying device 62, which are independently provided.
  • the processing device 60 of FIG. 7A includes a feed roller 42, a water bath 46, and a winder 44, which are sequentially arranged from the upstream side to the downstream side in the traveling direction of the fibroin fiber 70.
  • the fibroin fiber 70 sent out from the feed roller 42 is immersed in the water 47 in the water bath 46. As a result, the fibroin fiber 70 contracts.
  • the fibroin fiber 70 after the immersion is wound around the winder 44.
  • the feed roller 42 and the winder 44 may be omitted, and the fibroin fiber may be immersed in water in a so-called batch method.
  • the drying device 62 of FIG. 7B has a feed roller 42 and a winder 44, and a dry heat plate 64 arranged between them.
  • the fibroin fiber 70 unwound from the feed roller 43 is dried by traveling while being in contact with the dry heat surface 66 of the dry heat plate 64.
  • the fibroin fiber 70 can be further shrunk and the length of the fibroin fiber 70 can be unchanged. Drying by the drying device 62 is not essential.
  • Fibroin fiber (1) Modified fibroin A nucleic acid encoding a modified fibroin (PRT966) represented by SEQ ID NO: 40 was synthesized. An NdeI site was added to the 5'end and an EcoRI site was added downstream of the stop codon to the synthesized nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Then, the same nucleic acid was digested with NdeI and EcoRI and treated with a restriction enzyme. The excised nucleic acid was recombined into a protein expression vector pET-22b(+) to obtain an expression vector.
  • PRT966 modified fibroin represented by SEQ ID NO: 40
  • Escherichia coli BLR(DE3) was transformed with the obtained pET-22b(+) expression vector.
  • the transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 was 0.005.
  • the temperature of the culture solution was kept at 30° C., the flask culture was carried out for about 15 hours until the OD 600 reached 5, and a seed culture solution was obtained.
  • the seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD 600 was 0.05.
  • the temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min.
  • the temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Culturing was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration.
  • 1 M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce expression of the target modified fibroin.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • SDS-PAGE was performed using the cells prepared from the culture medium before and after the addition of IPTG, and the appearance of a band corresponding to the size of the target modified fibroin depending on the addition of IPTG resulted in the appearance of the target modified fibroin. The expression was confirmed.
  • the bacterial cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4).
  • the washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi).
  • the disrupted cells were centrifuged to obtain a precipitate.
  • the resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure.
  • the precipitate after washing was suspended in 8M guanidine buffer solution (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) at a concentration of 100 mg/mL, and the temperature was 60°C. The mixture was stirred for 30 minutes with a stirrer and dissolved. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation. Water was removed from the collected aggregated protein with a freeze dryer to obtain a freeze-dried powder of the target modified fibroin.
  • 8M guanidine buffer solution 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • the mixture was stirred for 30 minutes with a stirr
  • fibroin fiber 4.0% by mass of lithium chloride dissolved in dimethylsulfoxide (DMSO) was used as a solvent, and lyophilized powder of modified fibroin was dissolved therein. Then, insoluble matter and bubbles were removed to obtain a modified fibroin solution. Fibroin fibers were produced by dry and wet spinning using the obtained modified fibroin solution as a dope solution. The draw ratio was 6 times. A plurality of fibroin fibers each having a length of 30 cm were bundled to obtain a fiber bundle having a fineness of 150 denier. A 0.8 g lead weight was attached to each fiber bundle. In that state, the fiber bundle was immersed in water at 40° C. for 10 minutes, thereby contracting the fibroin fiber.
  • DMSO dimethylsulfoxide
  • the fiber bundle taken out from water was dried by leaving it at room temperature for 2 hours.
  • a fiber bundle of dried fibroin fibers containing the modified fibroin and having a single yarn diameter of 80 ⁇ m was obtained.
  • the obtained fiber bundle was subjected to the following fibroin fiber shape imparting test.
  • Shape imparting test comparative example As a cold perm liquid composed of the first liquid and the second liquid, "Volte" (trade name) manufactured by Melos Cosmetics Co., Ltd. was prepared. The fibroin fiber was wetted with water and wound around a metal cylindrical core material having a diameter of about 25 mm. The fibroin fiber wound around the core material was immersed in the first liquid of the cold perm solution for 1 hour, then washed with water and then immersed in the second solution of the cold perm solution for 1 hour. Finally, the fibroin fiber was washed with water. The fibroin fiber removed from the core had a curled shape. When the obtained fibroin fiber was washed once with water, the imparted shape almost disappeared.
  • Example 1 The same commercial cold perm solution as in the comparative example was used.
  • the fibroin fiber was wetted with water and wound around a metal cylindrical core material having a diameter of about 25 mm.
  • the fibroin fiber wrapped around the core material was immersed in an aqueous L-cysteine solution having a concentration of 14% by mass.
  • the L-cysteine aqueous solution was allowed to stand for 6 hours while being heated to 42°C.
  • the fibroin fiber wound around the core material is washed with water and then in a second liquid of cold perm solution at 42° C. for 3 hours and in a first liquid of cold perm solution at 42° C. for 3 hours. It was immersed in the second liquid of the above in order of 42° C. for 3 hours.
  • the fibroin fiber taken out from each liquid was washed with water and then immersed in the next liquid. After the final immersion in the second liquid, the fibroin fiber was washed with water.
  • the fibroin fiber removed from the core had a stronger curled shape than the comparative example.
  • the obtained fibroin fiber maintained the imparted shape to some extent even after being subjected to washing and drying three times.
  • Example 2 The first commercially available cold perm solution as in the comparative example was mixed with 10 parts by mass, 50 parts by mass of L-cysteine, and 40 parts by mass of water to prepare a first liquid for testing containing L-cysteine. Prepared.
  • the fibroin fiber was wetted with water and wound around a metal cylindrical core material having a diameter of about 25 mm.
  • the fibroin fiber wrapped around the core material was immersed in the first liquid for the test at room temperature (about 20° C.) overnight.
  • the fibroin fiber was washed with water and then immersed in a second liquid of the same cold perm liquid commercially available as in the comparative example at room temperature (about 20° C.)° C. for 4 hours.
  • FIG. 8 is a photograph of fibroin fibers after washing with water.
  • (A1) is the fibroin fiber of Example 2 after washing with water once
  • (a2) is the fibroin fiber of Comparative Example after washing with water once
  • (b1) is the fibroin fiber of Example 2 after washing with water 5 times. It is a fiber.
  • the fibroin fiber of Example 2 sufficiently maintained the curled shape even after washing with water 5 times.
  • Example 3 A fibroin fiber having a curled shape was obtained in the same manner as in Example 2 except that the immersion time in the first liquid was changed to 4 hours and the immersion time in the second liquid was changed to overnight. ..
  • FIG. 9 is a photograph of fibroin fiber after washing with water.
  • (A1) is the fibroin fiber of Example 2 after one water wash
  • (a2) is the fibroin fiber of Example 3 after one water wash
  • (b1) is the fibroin fiber of Example 2 after five water washes.
  • Fibroin fiber (b2) is the fibroin fiber of Example 3 after washing with water 5 times. It was confirmed that Example 2 in which the immersion time in the first liquid was longer than the immersion time in the second liquid was more excellent in shape retention.
  • Example 4 The same commercial cold perm solution as in the comparative example was used.
  • the fibroin fiber was wetted with water and wound around a metal cylindrical core material having a diameter of about 25 mm.
  • the fibroin fiber wrapped around the core material was immersed in an aqueous L-cysteine solution having a concentration of 1% by mass.
  • the L-cysteine aqueous solution was allowed to stand for 39 minutes while being heated to 42°C.
  • the fibroin fiber wrapped around the core material is washed with water and then in a second solution of cold perm solution at 42°C for 30 minutes, and in a first solution of cold perm solution at 42°C for 30 minutes. It was immersed in the second liquid of the above in order of 42° C. for 30 minutes.
  • the fibroin fiber taken out from each liquid was washed with water and then immersed in the next liquid. After the final immersion in the second liquid, the fibroin fiber was washed with water.
  • the fibroin fiber removed from the core had a stronger curled shape than the comparative example.
  • each fibroin fiber taken out from water was dried at room temperature for 2 hours with 0.8 g of lead weight attached. After drying, the length of each fibroin fiber (the length of the fibroin fiber when dried from the wet state) was measured. From the obtained measured values, the restoration rate, elongation rate and shrinkage rate C of each fibroin fiber were calculated according to the following equations (1), (4) and (5).
  • the fibroin fiber before being imparted with the shape and the fibroin fiber of each example provided with the curled shape have an elongation ratio of 2 to 17%, a shrinkage ratio C of 2 to 17%, and a shrinkage ratio of 95 to 100%.
  • the restoration rate was shown. That is, these fibroin fibers exhibited the property of elongating when brought into a wet state and contracting when dried from a wet state.

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Abstract

L'invention concerne : un procédé de production de fibres de cheveux synthétiques auxquelles est conférée une forme spécifiée ; et des fibres de cheveux synthétiques auxquelles est conférée une forme spécifiée, les fibres s'allongeant lorsqu'elles se trouvent dans un état humide et se rétractant lorsqu'elles sont séchées à partir d'un état humide. Ce procédé de production de fibres de cheveux synthétiques comprend : l'introduction de cystéine à la surface de fibres de fibroïne comprenant de la fibroïne modifiée ; et la mise en contact des fibres de fibroïne avec une solution de modelage froide dans un état dans lequel les fibres de fibroïne sont maintenues dans un état conforme à une forme spécifiée. La forme spécifiée comprend une portion incurvée, une portion linéaire ou les deux.
PCT/JP2020/003551 2019-01-31 2020-01-30 Procédé de production de fibres de cheveux synthétiques, procédé de production de cheveux synthétiques, fibres de cheveux synthétiques et cheveux synthétiques WO2020158900A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029804A (zh) * 2022-07-11 2022-09-09 青岛融美发制品集团有限公司 一种具有形状记忆功能发丝的制备方法

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CN113677838A (zh) 2019-01-31 2021-11-19 爱德兰丝株式会社 人造毛发用纤维、人造毛发、人造毛发用纤维的制造方法和人造毛发的制造方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS547009B2 (fr) * 1974-09-27 1979-04-03
JPH0678216B2 (ja) * 1987-06-30 1994-10-05 日華化学株式会社 酸性パ−マネントウェ−ブ用剤
JP2003146855A (ja) * 2001-08-28 2003-05-21 Toshiharu Fukai ウエーブ・カール用パーマ液
JP2004043356A (ja) * 2002-07-11 2004-02-12 Toshiharu Fukai ウエーブ・カール用パーマ液
WO2007094086A1 (fr) * 2006-02-14 2007-08-23 Aderans Holdings Co., Ltd. Perruque
JP2013163883A (ja) * 2012-02-13 2013-08-22 Artnature Co Ltd かつら用毛髪の製造方法、及び、かつらの製造方法
WO2017127940A1 (fr) * 2016-01-27 2017-08-03 Dalhousie University Protéines de soie aciniforme d'araignée artificielles, procédés de fabrication et utilisations

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS547009B2 (fr) * 1974-09-27 1979-04-03
JPH0678216B2 (ja) * 1987-06-30 1994-10-05 日華化学株式会社 酸性パ−マネントウェ−ブ用剤
JP2003146855A (ja) * 2001-08-28 2003-05-21 Toshiharu Fukai ウエーブ・カール用パーマ液
JP2004043356A (ja) * 2002-07-11 2004-02-12 Toshiharu Fukai ウエーブ・カール用パーマ液
WO2007094086A1 (fr) * 2006-02-14 2007-08-23 Aderans Holdings Co., Ltd. Perruque
JP2013163883A (ja) * 2012-02-13 2013-08-22 Artnature Co Ltd かつら用毛髪の製造方法、及び、かつらの製造方法
WO2017127940A1 (fr) * 2016-01-27 2017-08-03 Dalhousie University Protéines de soie aciniforme d'araignée artificielles, procédés de fabrication et utilisations

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029804A (zh) * 2022-07-11 2022-09-09 青岛融美发制品集团有限公司 一种具有形状记忆功能发丝的制备方法

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