WO2021065851A1 - Agent d'ajustement de la rétention d'eau pour cheveux artificiels et procédé d'ajustement de la rétention d'eau - Google Patents

Agent d'ajustement de la rétention d'eau pour cheveux artificiels et procédé d'ajustement de la rétention d'eau Download PDF

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WO2021065851A1
WO2021065851A1 PCT/JP2020/036775 JP2020036775W WO2021065851A1 WO 2021065851 A1 WO2021065851 A1 WO 2021065851A1 JP 2020036775 W JP2020036775 W JP 2020036775W WO 2021065851 A1 WO2021065851 A1 WO 2021065851A1
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amino acid
seq
sequence
modified fibroin
fibroin
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PCT/JP2020/036775
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Japanese (ja)
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翔太 冨樫
佑之介 安部
正敏 関
高橋　英樹
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Ｓｐｉｂｅｒ株式会社
株式会社アデランス
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Publication of WO2021065851A1 publication Critical patent/WO2021065851A1/fr

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    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41G—ARTIFICIAL FLOWERS; WIGS; MASKS; FEATHERS
    • A41G3/00—Wigs
    • 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

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  • the present invention relates to a water retention adjusting agent for artificial hair and a method for adjusting water retention.
  • Non-natural fibers are widely used as hair for wigs, hair for hair growth and hair substitutes for headdresses.
  • synthetic fibers such as nylon, acrylic or polyester, regenerated collagen fibers and the like are used, and there is an advantage that fibers having a certain quality can be stably supplied as compared with human hair. Has.
  • Patent Document 1 As artificial hair having enhanced water absorption, it has a plurality of recesses that open on the surface, and the depth of the recess is the maximum width in the direction in which the width at the opening portion of the recess is maximum.
  • a polyester-based artificial hair characterized by being deeper than the length of the hair is disclosed.
  • Patent Document 2 describes a treatment liquid containing a hydrolyzable organosilicon compound and a hydrolysis accelerator as a method for producing a novel water-resistant regenerated protein fiber improved by reducing the moisture absorption rate and / or the water absorption rate.
  • a method for producing a water-resistant regenerated protein fiber including a dipping step is disclosed.
  • An object of the present invention is to provide an artificial hair water retention adjusting agent capable of adjusting the water retention of artificial hair in a simple step, and a method capable of easily adjusting the water retention of artificial hair.
  • modified fibroin has a water retention property similar to that of human hair.
  • the present invention is based on this novel finding.
  • the present invention relates to, for example, the following inventions.
  • a water retention regulator for artificial hair containing modified fibroin as an active ingredient.
  • a method for adjusting the water retention of artificial hair which comprises a step of incorporating modified fibroin into the artificial hair raw material.
  • the present invention it is possible to provide a water retention adjusting agent for artificial hair, which can adjust the water retention of artificial hair in a simple step. Further, according to the present invention, it is possible to provide a method capable of easily adjusting the water retention of human hair.
  • the water retention is adjusted without impairing the biodegradability. can do.
  • the degree of water retention of artificial hair can also be adjusted by adjusting the amount of modified fibroin (active ingredient) contained in the artificial hair raw material.
  • the water retention regulator for artificial hair according to the present embodiment contains modified fibroin as an active ingredient.
  • Water retention refers to the property of maintaining a state of retaining water.
  • the water retention adjusting agent for artificial hair according to the present embodiment the water retention of artificial hair can be adjusted to a degree close to that of human hair by improving or reducing the water retention of artificial hair.
  • the water retention modifier for artificial hair according to the present embodiment utilizes the property of modified fibroin that it has water retention similar to that of human hair.
  • the modified fibroin according to the present embodiment is represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing a domain sequence.
  • the modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) 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 not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.
  • modified fibroin 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 fibroin having the same amino acid sequence as naturally occurring fibroin.
  • “Naturally derived fibroin” as used herein is also represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing the domain sequence to be used.
  • modified fibroin may be one in which the amino acid sequence of naturally-derived fibroin is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived). It may be an amino acid sequence modified by modifying the gene sequence of fibroin, or an artificially designed and synthesized product that does not depend 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 fibroin-specific crystalline region (typically corresponding to the (A) n motif of an amino acid sequence) and an amorphous region (typically to the REP of an amino acid sequence).
  • An amino acid sequence that produces (corresponding.)) which is represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif.
  • (A) n motif shows 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 of 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 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% (meaning that it is composed only of alanine residues).
  • a plurality of (A) n motifs present in the domain sequence may be composed of at least seven alanine residues only.
  • REP shows an amino acid sequence consisting of 2 to 200 amino acid residues.
  • REP may be an amino acid sequence composed of 10 to 200 amino acid residues, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160, or It may be an amino acid sequence composed of 10 to 180 amino acid residues.
  • m represents an integer of 2 to 300, 8 to 300, 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, It may be an integer of 20 to 140 or 20 to 120.
  • the plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • 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 to the cloned naturally occurring fibroin gene sequence. It can be obtained by modifying. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).
  • Naturally-derived 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. Yes, specifically, for example, fibroin produced by insects or arachnids.
  • fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pyrai, ⁇ ⁇ (Anteraea perni), and tussah. ), Silk moth (Samia cinthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama), etc. Hornet silk protein can be mentioned.
  • insect-produced fibroin include, for example, the silk moth fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • fibroin produced by arachnids include spider silk proteins produced by spiders belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders
  • Spiders belonging to the genus Pronus such as spiders, spiders belonging to the genus Trinofundamashi, such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, spiders, spiders, spiders, spiders, etc.
  • Spiders belonging to the genus Ordgarius such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders
  • Spider silk proteins produced by spiders belonging to the genus Cyclosa such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders.
  • Spiders belonging to the genus Tetragnatha such as Ashidaka spider and Urokoa shinagamo, spiders belonging to the genus White spider (genus Leucage), spiders belonging to the genus Leucage, spiders belonging to the genus Leucage, spiders belonging to the genus Leucage Spiders belonging to the genus Azumi (Menosira genus) such as spiders and spiders, spiders belonging to the genus Dyschiriognatha such as Himea shinagamo, spiders belonging to the genus Dyschiriognatha, spiders, spiders, spiders, sea urchins, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders,
  • spider silk protein examples include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag and the like.
  • spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank sequence number AAC4011 (amino acid sequence), U47856 (base sequence)) ), U37520 (base sequence)), major amplifier speedin 1 [derived from Laterectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk proteinaspirin Numbers AAL32472 (amino acid sequence), AF441245 (base sequence)), major protein speedin 1 [derived from Europe protein austral
  • fibroin whose sequence information is registered in NCBI GenBank can be mentioned.
  • sequence information registered in NCBI GenBank among the sequences containing INV as DIVISION, spidroin, complete, fibroin, "silk and protein", or “silk and protein” are described as keywords in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.
  • the modified fibroin according to the present embodiment may be modified silk fibroin (modified amino acid sequence of silk protein produced by spiders), or modified spider silk fibroin (spider silk protein produced by spiders). It may be a modified amino acid sequence).
  • modified fibroin examples include modified fibroin (first modified fibroin) derived from the large spitting tube bookmarker thread protein produced in the large bottle-shaped gland of spiders, and a domain sequence with a reduced content of glycine residues.
  • a modified fibroin having a reduced domain sequence can be mentioned.
  • the first modified fibroin examples include proteins containing a domain sequence represented by the formula 1: [(A) n motif-REP] m.
  • the number of amino acid residues of the (A) n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, and an integer of 10 to 20. Is even more preferable, an integer of 4 to 16 is even more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable.
  • the number of amino acid residues constituting REP in the formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues.
  • the total number of residues of glycine residue, serine residue and alanine residue contained in the amino acid sequence represented by the formula 1: [(A) n motif-REP] m is the amino acid residue. It is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number.
  • the first modified fibroin contains the unit of the amino acid sequence represented by the formula 1: [(A) n motif-REP] m , and the C-terminal sequence is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 or It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.
  • the amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C-terminal of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.
  • the amino acid sequence shown in (1-i) SEQ ID NO: 4 (recombinant spider silk protein ADF3 KaiLargeNRSH1), or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 and 90
  • the sequence identity is preferably 95% or more.
  • the amino acid sequence shown by SEQ ID NO: 4 is the first to the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 5) consisting of the start codon, His10 tag and HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal.
  • the 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue.
  • the amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in SEQ ID NO: 3.
  • the modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 4.
  • the second modified fibroin has an amino acid sequence whose 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 replaced with another amino acid residue as compared with naturally occurring fibroin. ..
  • the second modified fibroin has a domain sequence of GGX and GPGXX in REP as compared with naturally occurring fibroin (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine. In at least one motif sequence selected from), it has an amino acid sequence corresponding to at least one or a plurality of glycine residues in the motif sequence being replaced with another amino acid residue. You may.
  • the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more of the total motif sequence.
  • the second modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and is located closest to the C-terminal side of the domain sequence (A) from the n motif to the above domain sequence.
  • the total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence.
  • the number of alanine residues with respect to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).
  • the second modified fibroin is preferably 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 is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.
  • fibroin modified fibroin or naturally-derived fibroin
  • domain sequence represented by the formula 1: [(A) n motif-REP] m it is located most on the C-terminal side from the domain sequence (A) n.
  • the 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.
  • w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence.
  • z / w (%) can be calculated by dividing z by w.
  • z / w in naturally derived fibroin will be described.
  • 663 types of fibroin (of which 415 types of arachnid-derived fibroin were extracted) were extracted.
  • Naturally derived fibroin containing the domain sequence represented by the formula 1: [(A) n motif-REP] m and having an amino acid sequence consisting of GGX in fibroin of 6% or less among all the extracted fibroins.
  • z / w was calculated by the above-mentioned calculation method. The result is shown in FIG.
  • the horizontal axis of FIG. 2 indicates z / w (%), and the vertical axis indicates frequency.
  • the z / w in naturally-derived fibroin is less than 50.9% (the highest is 50.86%).
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. Is even more preferable, and 80% or more is even more preferable.
  • the upper limit of z / w is not particularly limited, but may be, for example, 95% or less.
  • the second modified fibroin is, for example, modified from the cloned naturally occurring fibroin gene sequence by substituting at least a part of the base sequence encoding the glycine residue to encode another amino acid residue.
  • one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be replaced so that z / w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment 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.
  • Insertion and / or modification of the amino acid sequence corresponding to the addition may be carried out.
  • the other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than the glycine residue, but is a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and methionine ( Hydrophobic amino acid residues such as M) residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S) ) Residues, hydrophilic amino acid residues such as lysine (K) residue and glutamate (E) residue are preferred, valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.
  • (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met) - contains an amino acid sequence represented by PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by (2-ii) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • Modified fibroin can be mentioned.
  • the modified fibroin of (2-i) will be described.
  • the amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin.
  • every two (A) n motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence is further before the C-terminal sequence.
  • One [(A) n motif-REP] is inserted in.
  • amino acid sequence shown in SEQ ID NO: 8 two alanine residues are inserted on the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added. It was replaced with a serine (S) residue, and some amino acids on the C-terminal side were deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 7.
  • the amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and His tag sequence are added to the C-terminal of the sequence obtained by repeating the above four times.
  • the value of z / w in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%.
  • the z / w values in the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 9 are 58.7%, respectively. It is 70.1%, 66.1% and 70.0%.
  • x / y in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 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 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) 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 is preferably 95% or more.
  • 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.
  • X indicates an amino acid residue other than glycine.
  • the second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization and the like of modified fibroin.
  • tag sequence examples include affinity tags that utilize specific affinity (binding, affinity) with other molecules.
  • affinity tag a histidine tag (His tag) can be mentioned.
  • His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chromatography) is performed. Can be used for.
  • Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).
  • tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can also be used.
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • an "epitope tag” utilizing an antigen-antibody reaction can also be used.
  • a peptide (epitope) exhibiting antigenicity as a tag sequence an antibody against the epitope can be bound.
  • the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like.
  • a tag sequence in which the tag sequence can be separated by a specific protease can also be used.
  • the modified fibroin from which the tag sequence has been separated can also be recovered.
  • modified fibroin containing the tag sequence the amino acids represented by (2-iii) SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799).
  • modified fibroins comprising the sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (2-iv) SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..
  • amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • the amino acid sequence shown by SEQ ID NO: 11 (including His tag sequence and hinge sequence) is added to the N-terminal of the indicated amino acid sequence.
  • the modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 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 the domain sequence represented by the formula 1: [(A) n motif-REP] m.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP.
  • X indicates an amino acid residue other than glycine.
  • 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 secretory signal can be appropriately set according to the type of host.
  • the third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) n motif as compared with 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 more (A) n motifs as compared with the naturally occurring fibroin.
  • the third modified fibroin may have an amino acid sequence corresponding to a 10-40% deletion of the (A) n motif from naturally occurring fibroin.
  • 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 It may have an amino acid sequence corresponding to the deletion of.
  • the third modified fibroin has a domain sequence of at least two consecutive (A) n- motif deletions and one (A) from the N-terminal side to the C-terminal side as compared to naturally occurring fibroin. ) It may have an amino acid sequence corresponding to the deletion of the n-motif being repeated in this order.
  • the third modified fibroin may have an amino acid sequence corresponding to the deletion of the (A) n motif at least every other two domain sequences from the N-terminal side to the C-terminal side. ..
  • the third modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N-terminal side to the C-terminal side.
  • -REP The number of amino acid residues in the REP of the unit is sequentially compared, and when the number of amino acid residues in the REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to When x is the maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units, which is 11.3, and y is the total number of amino acid residues in the domain sequence.
  • the number of alanine residues with respect to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (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 arrangement called n motifs.
  • Two adjacent [(A) n motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, there may be a [(A) n motif-REP] unit that is not selected.
  • pattern 1 (comparison between the first REP and the second REP and comparison between the third REP and the fourth REP)
  • pattern 2 (comparison between the first REP and the second REP, and a comparison).
  • 4th REP and 5th REP comparison Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison
  • Pattern 4 (1st REP and (Comparison of the second REP) is shown. There are other selection methods.
  • the number of amino acid residues of each REP in two adjacent [(A) n motif-REP] units selected is compared.
  • the comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1.
  • each pattern add up the total number of amino acid residues of the two adjacent [(A) n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of (A) n motif. is there.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximized is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
  • x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.
  • x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% or more, and particularly preferably 80% or more.
  • the upper limit of x / y is not particularly limited and may be, for example, 100% or less.
  • x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x.
  • / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.
  • the third modified fibroin is a modified fibroin in which at least 7 of the (A) n motifs present in the domain sequence are composed of only alanine residues
  • the x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, 70% or more is even more preferable, and 80% or more. It is particularly preferable to have.
  • the upper limit of x / y is not particularly limited and may be 100% or less.
  • the horizontal axis of FIG. 3 indicates x / y (%), and the vertical axis indicates frequency.
  • the x / y of naturally occurring fibroin is less than 64.2% (the highest is 64.14%).
  • the third modified fibroin deletes one or more of the sequences encoding the (A) n motif from the cloned naturally occurring fibroin gene sequence so that x / y is 64.2% or more.
  • an amino acid sequence corresponding to the deletion of one or more (A) n motifs so that x / y is 64.2% or more is designed and designed from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to the above may be modified.
  • 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) contains an amino acid sequence represented by PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • Modified fibroin can be mentioned.
  • the modified fibroin of (3-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 17 is from the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin, every other (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 sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.
  • the value of x / y in the jagged ratio of 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 is 15.0%.
  • the value of x / y in the amino acid sequence shown in SEQ ID NO: 17 and the amino acid sequence shown in SEQ ID NO: 7 is 93.4%.
  • the value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%.
  • the value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.8%.
  • the modified fibroin of (3-i) may consist of 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) 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 is preferably 95% or more.
  • 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 is N-terminal to C-terminal.
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other
  • x / y is preferably 64.2% or more.
  • the third modified fibroin may contain the tag sequence described above at either or both of the N-terminus and the C-terminus.
  • modified fibroin containing the tag sequence the amino acids represented by (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799).
  • modified fibroins comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (3-iv) SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..
  • amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are the N-terminals of the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • the amino acid sequence shown by (including His tag sequence and hinge sequence) is added.
  • the modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 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 the domain sequence represented by the formula 1: [(A) n motif-REP] m.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (3-iv) has 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, and is N-terminal to C-terminal.
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other Let x be the maximum value of the total value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues in REP is 1.8 to 11.3.
  • x / y is preferably 64.2% or more.
  • 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 secretory signal can be appropriately set according to the type of host.
  • the fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) n motifs and a reduced content of glycine residues as compared with naturally occurring fibroin.
  • the domain sequence of the fourth modified fibroin lacked at least one or more (A) n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the above-mentioned second modified fibroin and the third modified fibroin. Specific aspects and the like are as described in the second modified fibroin and the third modified fibroin.
  • the fourth modified fibroin (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) ), The amino acid sequence represented by 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
  • modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by Specific embodiments of the modified fibroin comprising the amino acid sequence set forth 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 had its domain sequence replaced with one or more amino acid residues in the REP compared to naturally occurring fibroin, and / or REP. It may have an amino acid sequence containing a region having a large hydrophobic index locally, which corresponds to the insertion of one or a plurality of amino acid residues having a large hydrophobic index.
  • the region having a locally large hydrophobicity index is preferably composed of consecutive 2 to 4 amino acid residues.
  • the 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 is more preferably a residue.
  • one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index as compared with naturally occurring fibroin, and / or one or more amino acid residues in REP.
  • one or more amino acid residues were substituted, deleted, inserted and / or added as compared with naturally occurring fibroin.
  • the fifth modified fibroin leaves one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting for a group (eg, an amino acid residue with a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in the REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the REP.
  • a group eg, an amino acid residue with a positive hydrophobicity index
  • one or more hydrophilic amino acid residues in the REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the REP.
  • an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence In each case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acids in the REP.
  • the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.
  • 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-terminal side to the C-terminal 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 consecutive 4 amino acid residues is 2.6 or more is defined as p.
  • hydrophobicity index of amino acid residues
  • a known index Kyte J, & Doolittle R (1982) "A simple method for dispensing the hydropathic character of Protein. 105-132
  • HI hydrophobicity index
  • sequence A [(A) n motif-REP] m.
  • sequence A the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) n motif-REP] m.
  • sequence A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated for all REPs contained in the sequence A.
  • the average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues).
  • the average value of the hydrophobicity index is obtained for all consecutive 4 amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times).
  • a region in which the average value of the hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index", it should be included as one amino acid residue in the region. become.
  • the total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in the sequence A is q.
  • the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.
  • p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more.
  • the upper limit of p / q is not particularly limited, but may be, for example, 45% or less.
  • the fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (eg, a hydrophobic index) in the REP so that the amino acid sequence of the cloned naturally occurring fibroin satisfies the above p / q condition.
  • Amino acid residue with a negative value 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 in the REP.
  • a hydrophobic amino acid residue for example, an amino acid residue with a positive hydrophobicity index
  • an amino acid sequence satisfying the above p / q condition 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 in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring fibroin, and / or one or more in the REP.
  • the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. ..
  • the amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) Is preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.
  • the fifth modified fibroin (5-i) the amino acid sequence set forth in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666).
  • a modified fibroin containing (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can be mentioned.
  • the modified fibroin of (5-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 19 consists of 3 amino acid residues every other REP, except for the domain sequence of the terminal on the C-terminal side, with respect to the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410).
  • Two amino acid sequences (VLI) are inserted, a part of glutamine (Q) residue is replaced with a serine (S) residue, and a part of amino acid on the C-terminal side is deleted.
  • the amino acid sequence shown by SEQ ID NO: 20 is the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. is there.
  • the amino acid sequence shown in SEQ ID NO: 21 is the amino acid sequence shown in SEQ ID NO: 8 with two amino acid sequences (VLI) consisting of three amino acid residues inserted every other REP.
  • the modified fibroin of (5-i) may consist of 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) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 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 is preferably 95% or more.
  • 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 most on the C-terminal side (A) n.
  • P / q is preferably 6.2% or more.
  • the fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.
  • modified fibroin containing a tag sequence the amino acid sequence set forth in (5-iii) 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 set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 can be mentioned.
  • amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences shown in SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including array and hinge array) is added.
  • the modified fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) is also a protein containing the domain sequence represented by the formula 1: [(A) n motif-REP] m.
  • the sequence identity is preferably 95% or more.
  • 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 most on the C-terminal side (A) n.
  • P / q is preferably 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 secretory signal can be appropriately set according to 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 preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.
  • the content of the GPGXXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more.
  • the upper limit of the GPGXX motif content is not particularly limited and may be 50% or less, or 30% or less.
  • GPGXX motif content is a value calculated by the following method.
  • Formula 1 [(A) n- motif-REP] m
  • Formula 2 [(A) n- motif-REP] m-
  • s be the number obtained by multiplying the total number by 3 (that is, corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence.
  • the GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluding (A) n motifs.
  • the sequence obtained by excluding the sequence from the (A) n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is targeted at "the most C-terminal side".
  • the sequence from the n motif to the C-terminal of the domain sequence may contain 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, and this effect is eliminated.
  • the "GPGXX motif” is located at the C-terminal of the REP, even if "XX" is, for example, "AA”, it is treated as a "GPGXX motif".
  • FIG. 5 is a schematic diagram showing the domain sequence of modified fibroin.
  • the sixth modified fibroin has a glutamine residue content of preferably 9% or less, more preferably 7% or less, further preferably 4% or less, and particularly preferably 0%. ..
  • the "glutamine residue content” is a value calculated by the following method.
  • Formula 1 [(A) n- motif-REP] m
  • Formula 2 [(A) n- motif-REP] m-
  • A) Fibroin containing a domain sequence represented by n- motif (modified fibroin or naturally derived) In fibroin) all the sequences from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 5).
  • the total number of glutamine residues contained in the region is u
  • the sequence from the (A) n motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) n.
  • the glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif.
  • the reason why "the sequence from the (A) n motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.
  • the sixth modified fibroin corresponds to its domain sequence being deleted from one or more glutamine residues in the REP or replaced with other amino acid residues as compared to 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 is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue.
  • the hydrophobicity index of amino acid residues is as shown in Table 1.
  • amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M).
  • Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can.
  • amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferable.
  • the sixth modified fibroin has a REP hydrophobicity of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable.
  • the upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.
  • the "hydrophobicity of REP” is a value calculated by the following method.
  • Formula 1 [(A) n- motif-REP] m
  • Formula 2 [(A) n- motif-REP] m-
  • all the sequences from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A” in FIG. 5).
  • the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the sequence from the (A) n motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the n motif.
  • the reason for targeting is the above-mentioned reason. The same is true.
  • the sixth modified fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in REP.
  • modification corresponding to the substitution of one or more amino acid residues there may be further 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 deletes one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and / or removes one or more glutamine residues in REP. It can be obtained by substituting with the amino acid residue of. Also, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring 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 this 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 SEQ ID NO: Modified fibroin containing the amino acid sequence represented by No.
  • the modified fibroin of (6-i) will be described.
  • the amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting VL for all QQs in the amino acid sequence (Met-PRT410) shown in SEQ ID NO: 7.
  • the amino acid sequence shown in SEQ ID NO: 26 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with TS, and the remaining Qs are replaced with A.
  • the amino acid sequence shown in SEQ ID NO: 27 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VL, and the remaining Qs are replaced with I.
  • the amino acid sequence shown in SEQ ID NO: 28 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VI, and the remaining Qs are replaced with L.
  • the amino acid sequence shown in SEQ ID NO: 29 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VF, and the remaining Qs are replaced with I.
  • the amino acid sequence shown in SEQ ID NO: 30 is obtained by substituting VL for all QQs in the amino acid sequence (Met-PRT525) shown in SEQ ID NO: 8.
  • the amino acid sequence shown in SEQ ID NO: 31 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.
  • 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 residual glutamine.
  • the group content is 9% or less (Table 2).
  • the modified fibroin of (6-i) has 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 indicated amino acid sequence.
  • the modified fibroins of (6-ii) are 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 or SEQ ID NO: 42. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence.
  • the modified fibroin of (6-ii) is also a domain represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.
  • the modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-ii) preferably has 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, visualization and the like of modified fibroin.
  • modified fibroin containing the tag sequence (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 (PRT918), Modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028), or (PRT1028).
  • amino acid sequences shown by 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 shown by SEQ ID NO: 11 at the N-terminal of the amino acid sequence.
  • the amino acid sequence (including His tag sequence and hinge sequence) is added.
  • 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 fibroins of (6-iii) are 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 or SEQ ID NO: 44. It may consist of the indicated amino acid sequence.
  • the modified fibroins of (6-iv) are 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 or SEQ ID NO: 44. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence.
  • the modified fibroin of (6-iv) is also a domain represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.
  • the modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-iv) preferably has 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 secretory signal can be appropriately set according to 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 above-mentioned characteristics.
  • the modified fibroin may be hydrophilic modified fibroin or hydrophobic modified fibroin.
  • hydrophilic modified fibroin is a value obtained by obtaining the total hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then dividing the total by the total number of amino acid residues. It is a modified fibroin having (average HI) of 0 or less.
  • the hydrophobicity index is as shown in Table 1.
  • modified fibroin having an average HI of more than 0 may be referred to as hydrophobic modified fibroin.
  • hydrophilic modified fibroin examples include the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, and SEQ ID NO: 14. Or the amino acid sequence shown by SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 7, amino acid sequence shown by SEQ ID NO: 8 or SEQ ID NO: 9, amino acid represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.
  • modified fibroins comprising the amino acid sequence set forth in the sequence, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • hydrophobically modified fibroin examples include the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43, SEQ ID NO: 35.
  • modified fibroins comprising the amino acid sequences set forth in 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.
  • the modified fibroin is preferably a modified fibroin having an average value (mean HI) of the hydrophobicity index of more than ⁇ 0.8 from the viewpoint of having water retention closer to that of human hair.
  • the average HI of the modified fibroin is, for example, -0.7 or higher, -0.6 or higher, -0.5 or higher, -0.4 or higher, -0.3 or higher, -0.2 or higher, -0.1 or higher, It may be 0 or more, 0.1 or more, 0.2 or more, or 0.3 or more, and may be 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, 1. It may be 0 or less, or 0.9 or less.
  • the modified fibroin having an average HI of more than ⁇ 0.8 may be, for example, the hydrophobic modified fibroin described above, or may be a hydrophilic modified fibroin having an average HI of more than ⁇ 0.8.
  • Modified fibroins having an average HI of more than ⁇ 0.8 include, for example, the fifth fibroin, SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), and the sixth fibroin.
  • SEQ ID NO: 33 PRT888
  • SEQ ID NO: 34 PRT965
  • SEQ ID NO: 35 PRT888
  • SEQ ID NO: 36 PRT916
  • SEQ ID NO: 37 PRT918
  • SEQ ID NO: 38 SEQ ID NO: 38
  • SEQ ID NO: 39 SEQ ID NO: 39
  • SEQ ID NO: 39 SEQ ID NO: 39 (PRT698)
  • SEQ ID NO: 33 PRT888
  • SEQ ID NO: 34 PRT965
  • SEQ ID NO: 35 PRT888
  • SEQ ID NO: 36 PRT916
  • SEQ ID NO: 37 PRT918)
  • SEQ ID NO: 38 SEQ ID NO: 38
  • SEQ ID NO: 39 SEQ ID NO: 39
  • the modified fibroin according to the present embodiment can be produced by a conventional method using a nucleic acid encoding the modified fibroin.
  • the nucleic acid encoding the modified fibroin may be chemically synthesized based on the base sequence information, or may be synthesized by using a PCR method or the like.
  • the produced modified fibroin can be isolated and purified by a commonly used method.
  • the water retention regulator for artificial hair may contain one modified fibroin alone, or a combination of two or more modified fibroins. It may be.
  • the water retention adjusting agent for artificial hair may further contain other additives (ingredients other than the active ingredient) depending on its form, use and the like.
  • Additives include, for example, plasticizers, leveling agents, cross-linking agents, crystal nucleating agents, antioxidants, UV absorbers, colorants, fillers, and synthetic resins.
  • the content of the additive may be 50 parts by mass or less with respect to 100 parts by mass of the total amount of the water retention adjusting agent for artificial hair.
  • the water-retaining agent for artificial hair according to the present embodiment may be in any form of, for example, powder, paste, liquid (for example, suspension, solution), or pellet.
  • the water retention modifier for artificial hair according to the present embodiment may also be in the form of a molded product such as a fiber, a film, a gel, a porous body, or particles.
  • the form of the water retention adjusting agent for artificial hair according to the present embodiment may be appropriately set according to the use of the artificial hair and the like.
  • the water-retaining agent for artificial hair contains modified fibroin as a main component, it can be molded into any of the above-mentioned forms.
  • the molded product may be a molded product obtained by molding the modified fibroin itself, or may be molded by combining the modified fibroin with another material.
  • the protein obtained by the above-mentioned method for producing modified fibroin may be dried and made into powder.
  • the protein powder may contain other additives, if desired.
  • the water retention regulator for artificial hair according to the present embodiment is prepared in the form of a liquid (for example, a solution), for example, the protein obtained by the above-mentioned method for producing modified fibroin is dissolved in a solvent capable of dissolving the modified fibroin. It may be made liquid (modified fibroin solution).
  • the modified fibroin solution may contain other additives, if desired.
  • the solvent capable of dissolving the modified fibroin include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, hexafluoroisopropanol (HFIP) and the like.
  • An inorganic salt may be added to the solvent as a dissolution accelerator.
  • the water retention conditioner for artificial hair according to the present embodiment is prepared in the form of fibers
  • the above-mentioned modified fibroin solution is used as a dope solution, and known spinning such as wet spinning, dry spinning, dry wet spinning or melt spinning is used. It may be spun by a method into fibers (modified fibroin fibers).
  • the form of the fiber may be a single yarn, a composite yarn such as a blended yarn, a mixed fiber yarn, a mixed weaving yarn, a mixed weaving yarn, a twisted yarn and a covering yarn, or a non-woven fabric or the like. Good.
  • the modified fibroin fiber may be a short fiber or a long fiber.
  • the modified fibroin fiber may be the modified fibroin fiber alone or may be combined with other fibers. That is, a single yarn composed of only modified fibroin fibers and a composite yarn composed of a combination of modified fibroin fibers and other fibers may be used alone or in combination thereof.
  • the single yarn and the composite yarn may be spun yarns in which short fibers are twisted, or filament yarns in which long fibers are twisted or not twisted.
  • the modified fibroin fiber whether it is a short fiber or a long fiber, can be used as a fiber alone or in combination with other fibers without being processed into a yarn.
  • the content of the modified fibroin fiber is preferably 20% by mass or more, more preferably 30% by mass or more, and more preferably 40% by mass, based on the total amount of fibers. It is more preferably more than that, and even more preferably 50% by mass or more.
  • the water-retaining agent for artificial hair according to the present embodiment is prepared in the form of a film, gel, porous body, particles, etc., for example, Japanese Patent Application Laid-Open No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735. It can be manufactured according to the method described in the publications and the like.
  • the method for adjusting the water retention of artificial hair according to the present embodiment includes a step of incorporating modified fibroin into the artificial hair raw material. Since the modified fibroin according to the present invention has excellent water retention or has water retention close to that of human hair, the water retention of artificial hair can be adjusted by incorporating it into the artificial hair raw material.
  • the artificial hair raw material is not particularly limited as long as it is used as a raw material for artificial hair.
  • the artificial hair raw material may be, for example, fibers, a dope solution capable of forming fibers by spinning, resin pellets capable of forming fibers by spinning, or the like.
  • the step of containing the modified fibroin may be a step of containing the modified fibroin by incorporating the above-mentioned water retention adjusting agent for artificial hair according to the present invention.
  • the modified fibroin may be contained in the artificial hair raw material in the form of a molded body such as long fibers, short fibers, gels, particles, porous bodies, films, etc., and the artificial hair raw material is coated with liquid modified fibroin. Therefore, it may be contained in the artificial hair raw material.
  • the liquid modified fibroin may be, for example, a liquid composition containing the modified fibroin and a solvent.
  • the method for containing the modified fibroin is not particularly limited, and a method of mixing the modified fibroin fiber (water retention modifier for artificial hair) with the artificial hair raw material fiber may be used, and the modified fibroin is prepared in the form of the above-mentioned molded product.
  • a method of forming artificial hair by combining an artificial hair water retention adjusting agent (modified fibroin) with an artificial hair raw material may be used, and the modified fibroin (modified fibroin) is applied to artificial hair in the above-mentioned liquid form. It may be a method of coating or spray-coating the raw material. Further, it may be a method of forming artificial hair by molding the modified fibroin (which may contain other additives if necessary) itself.
  • a powdery water retention regulator for artificial hair is added to a soluble solvent.
  • the dope solution may be spun to form artificial hair.
  • the doping solution may contain other artificial hair raw materials such as regenerated collagen.
  • the artificial hair raw material may be mixed with a powdery or pelletized water retention conditioner for artificial hair (modified fibroin) and melt-spun to form artificial hair.
  • the content of the modified fibroin in the artificial hair is preferably 20% by mass or more, more preferably 30% by mass or more, further preferably 40% by mass or more, more preferably 50% by mass or more, based on the total amount of the artificial hair. It is even more preferably mass% or more.
  • the upper limit of the content of the modified fibroin may be 100% by mass or 90% by mass or less based on the total amount of artificial hair.
  • the amino acid sequence shown in SEQ ID NO: 40 is a sequence in which the regions of 20 domain sequences existing in the amino acid sequence (Met-PRT410) shown in SEQ ID NO: 7 are repeated twice for the purpose of improving the hydrophobicity.
  • the amino acid sequence shown by SEQ ID NO: 11 is further added to the N-terminal to which all QQs are replaced with VF and the remaining Qs are replaced with I (amino acid sequence shown by SEQ ID NO: 32).
  • the average (mean HI) of the hydrophobicity index of modified fibroin PRT966 is 0.466.
  • nucleic acid encoding the designed modified fibroin (modified spider silk fibroin) PRT966 was synthesized.
  • An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon.
  • the nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • OD 600 was added to the seed culture medium in a jar fermenter to which 500 mL of the production medium (Table 5) was added. Was added so as to be 0.05.
  • the temperature of the culture solution was maintained at 37 ° C., and the cells were cultured under constant pH 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 cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of modified fibroin.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL, and the temperature was 60 ° C.
  • 8M guanidine buffer 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • the white agglutinated protein obtained after dialysis was recovered by centrifugation, water was removed by a freeze-dryer, and the freeze-dried powder was recovered to obtain modified fibroin (PRT966).
  • modified fibroin fiber modified spider silk fibroin fiber
  • the fiber diameter of the fiber containing the modified fibroin PRT966 was 99 ⁇ m.
  • Spinning conditions Spinning nozzle hole diameter: 0.3 mm
  • Coagulant 18% by mass sodium sulfate aqueous solution
  • Coagulant temperature 40 ° C
  • the water retention of the modified fibroin fiber and the above fiber was evaluated by the following procedures (1) to (7).
  • Each fiber was cut into a length of 15 cm.
  • (2) Calculate the value of the weight per fiber and the side area from the weight per 50 fibers, adjust the number of fibers so that the sum of the side areas of the fiber bundles is 500 cm 2, and prepare each sample. Made.
  • the side area was calculated on the assumption that the fiber diameter was measured and the fiber cross section was circular.
  • Each sample was allowed to stand for 24 hours or more in a constant temperature and humidity chamber set at a temperature of 20 ° C. and a relative humidity of 65%.
  • the weight of each sample after curing (standing) was measured and used as the initial weight ( Wi ).
  • Each sample was impregnated in tap water at 25 ° C. for 15 minutes, then divided into small portions in a washing net and dehydrated in a washing machine for 15 minutes.
  • each sample is allowed to stand in a constant temperature and humidity chamber set to a temperature of 20 ° C. and a relative humidity of 65%, and then each sample is placed every hour thereafter. Weight (W) was measured for a total of 8 hours.
  • the water content was calculated from the weight of each sample at each time, and the water retention was evaluated.
  • the water content [g] shown in Table 6 and the ratio [%] of the water content per fiber weight shown in Table 7 and FIG. 6 were calculated from the following formulas (a) and (b).
  • Formula (b): Ratio of water content per fiber weight [%] Water content [G] / Initial weight of sample ( Wi ) [g] x 100
  • the regenerated collagen fiber (Comparative Example 2) has an initial water retention amount (0 hours) and a water retention capacity (water retention amount equivalent to that of human hair) which are too high as compared with human hair. It takes 4 to 6 hours to become). It was confirmed that the polyester fibers (Comparative Examples 3 and 4) were significantly inferior in both the initial water retention amount and the water retention capacity as compared with human hair. On the other hand, it was confirmed that the water retention of the fiber containing the modified fibroin (Example 1) was very close to that of human hair (Comparative Example 1) in terms of both the initial water retention amount and the water retention capacity, and the fiber was modified. It has been shown that fibroin is suitable as a water retention regulator for artificial hair.
  • the modified fibroin according to the present invention with regenerated collagen as a water retention regulator, the water retention of human hair can be approached.

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Abstract

La présente invention aborde le problème de la réalisation d'un agent d'ajustement de la rétention d'eau qui est capable d'ajuster facilement la capacité de rétention d'eau de cheveux artificiels par un procédé simple ; et d'un procédé qui est capable d'ajuster facilement la capacité de rétention d'eau de cheveux artificiels. La solution selon l'invention porte sur un agent d'ajustement de la rétention d'eau qui contient, en tant que principe actif, une fibroïne modifiée ; et sur un procédé d'ajustement de la capacité de rétention d'eau de cheveux artificiels, ledit procédé comprenant une étape d'obtention d'un matériel de départ de cheveux artificiels contenant une fibroïne modifiée.
PCT/JP2020/036775 2019-09-30 2020-09-29 Agent d'ajustement de la rétention d'eau pour cheveux artificiels et procédé d'ajustement de la rétention d'eau WO2021065851A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014002605A1 (fr) * 2012-06-28 2014-01-03 スパイバー株式会社 Fibre de protéine teintée dans la masse et procédé pour produire celle-ci
WO2019151424A1 (fr) * 2018-01-31 2019-08-08 Ｓｐｉｂｅｒ株式会社 Composition de fibroïne, solution de fibroïne, et procédé de fabrication de fibres de fibroïne
WO2020022395A1 (fr) * 2018-07-25 2020-01-30 Ｓｐｉｂｅｒ株式会社 Fibre de cheveux synthétiques, procédé de fabrication associé, et cheveux synthétiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014002605A1 (fr) * 2012-06-28 2014-01-03 スパイバー株式会社 Fibre de protéine teintée dans la masse et procédé pour produire celle-ci
WO2019151424A1 (fr) * 2018-01-31 2019-08-08 Ｓｐｉｂｅｒ株式会社 Composition de fibroïne, solution de fibroïne, et procédé de fabrication de fibres de fibroïne
WO2020022395A1 (fr) * 2018-07-25 2020-01-30 Ｓｐｉｂｅｒ株式会社 Fibre de cheveux synthétiques, procédé de fabrication associé, et cheveux synthétiques

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