WO2018164190A1 - Fibres de fibroïne synthétique - Google Patents

Fibres de fibroïne synthétique Download PDF

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WO2018164190A1
WO2018164190A1 PCT/JP2018/008800 JP2018008800W WO2018164190A1 WO 2018164190 A1 WO2018164190 A1 WO 2018164190A1 JP 2018008800 W JP2018008800 W JP 2018008800W WO 2018164190 A1 WO2018164190 A1 WO 2018164190A1
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amino acid
seq
fibroin
acid sequence
sequence
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PCT/JP2018/008800
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Japanese (ja)
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潤一 菅原
森田　啓介
中村　浩之
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Ｓｐｉｂｅｒ株式会社
小島プレス工業株式会社
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Publication of WO2018164190A1 publication Critical patent/WO2018164190A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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 an artificial fibroin fiber.
  • Silk which is a kind of natural fibroin fiber, is known as a fiber that sufficiently satisfies the touch and has a high-quality feeling.
  • fibers used in clothing, bedding, etc. are required to have softness and heat retention. Therefore, silk used for clothing, bedding, and the like is subjected to, for example, shrink processing to increase bulkiness, thereby imparting flexibility and heat retention in some cases.
  • synthetic fibers such as polyester fibers, polyamide fibers, and acrylic fibers are generally used for clothing, bedding, etc., and these synthetic fibers have a shrinkage ratio of 40% or more when brought into contact with boiling water.
  • An object of the present invention is to provide an artificial fibroin fiber that has a sufficiently high shrinkage rate and can be manufactured safely.
  • the present invention relates to the following inventions, for example.
  • Shrinkage rate ⁇ 1 ⁇ (length of artificial fibroin fiber subjected to shrinking process including contact with water having less than boiling point / length of artificial fibroin fiber before performing the shrinking process) ⁇ ⁇ 100 (%)
  • the artificial fibroin fiber according to [1] wherein the modified fibroin is a modified spider silk fibroin.
  • an artificial fibroin fiber that has a sufficiently high shrinkage rate and can be manufactured safely.
  • the modified fibroin according to the present embodiment has a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing.
  • an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either one or both of the N-terminal side and the C-terminal side of the domain sequence.
  • the N-terminal sequence and the C-terminal sequence are not limited to these, but are typically regions having no amino acid motif repeat characteristic of fibroin and consisting of about 100 amino acids.
  • modified fibroin means an 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 a fibroin having the same amino acid sequence as that of naturally occurring fibroin.
  • Natural fibroin as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif.
  • a protein comprising a domain sequence to be processed.
  • the amino acid sequence of naturally-occurring fibroin may be used as it is, depending on the amino acid sequence of naturally-occurring fibroin.
  • the amino acid sequence may be modified (for example, the amino acid sequence may be modified by modifying the gene sequence of the cloned natural fibroin), and may be artificially designed and used independently of the natural fibroin. It may be synthesized (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).
  • domain sequence refers to a fibroin-specific crystal region (typically corresponding to the (A) n motif in the amino acid sequence) and an amorphous region (typically in the REP of the amino acid sequence).
  • (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 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 (A) n motif may be 40% or more, such as 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 alone.
  • 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.
  • m represents an integer of 2 to 300, and may be an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • (A) n motif is preferably alanine residues is 83% or more to the total number of amino acid residues in the n motif, more preferably 86% or more, 90% or more Is more preferably 95% or more, particularly preferably 100% (meaning that it is composed of only alanine residues). It is preferable that at least seven of the (A) n motifs present in the domain sequence are composed of only alanine residues. Consists of only alanine residues means that (A) n motif is (A) n (A represents an alanine residue, n is an integer of 2 to 20, preferably 4 to 20, more preferably 4 to It represents an integer of 16).
  • 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 a plurality of amino acid residues to the cloned gene sequence of naturally derived fibroin. It can be obtained by modifying the above. 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-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
  • Naturally-derived 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.
  • Specific examples include fibroin produced by insects or spiders.
  • fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandaraina), Tengea (Antherea yamanai), ⁇ ⁇ (Antereaperyi), ⁇ ⁇ (Eriothyraminey) ), Silkworms (Samia cythia), chestnut worms (Caligula japonica), Chussa moth (Anthereaea mylitta), silkworms produced by silkworms such as Antheraea assamata, vespasam worms Examples include silk proteins.
  • fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).
  • Fibroin produced by spiders includes, for example, spiders belonging to the genus spider (Araneus spp.) Such as the spider spider, the spider spider, the red spider spider, and the bean spider, the genus spiders of the genus Araneus, the spider spider spider, the spider spider genus e Spiders, spiders such as spiders, spiders belonging to the genus Spider, spiders belonging to the genus Pronos, spiders belonging to the genus Trinofunda, such as Torinofundamas (genus Cyrtarachne) Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Spider (Ordgarius genus), such as the spiders, the spiders, and the spiders belonging to the genus Ordgarius Spiders belonging to the genus Argiope, such as the genus Argiope, spiders belonging to the genus Arachnura, such as the white-tailed spider, spiders belonging to the
  • Spiders belonging to the genus Azumigumi (Menosira), spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the common spider spider, the black spider spider, the genus Spider genus belonging to the genus Spider belonging to the genus (L) and the genus Spider belonging to the genus (L) Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops
  • Examples include spider silk protein.
  • the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.
  • fibroin produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin- 4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidolin 1 [derived from Nephila clavipes (GenBank accession number AAC4, amino acid sequence A04) U37520 (base sequence)), major sample spidroin 1 [La rodectus hesperus origin] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Nephila clavata origin] (GenBank accession number AAL32447, amino acid sequence 45, amino acid sequence) major amploidate
  • Naturally derived fibroin include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidin, sample, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION from sequences including INV as DIVISION among the sequence information registered in NCBI GenBank. It can be confirmed by extracting a character string of a specific product from the sequence, CDS, and a sequence in which the specific character string is described from SOURCE to TISSUE TYPE.
  • the modified fibroin according to the present embodiment may be a modified silk fibroin (a modified amino acid sequence of a silk protein produced by a silkworm), and a modified spider silk fibroin (an amino acid sequence of a spider silk protein produced by a spider). It may be modified).
  • the modified fibroin with a reduced content of glycine residues (modified fibroin according to the first embodiment), (A) the content of the n motif is reduced.
  • the modified fibroin according to the first embodiment has an amino acid sequence in which the content of a glycine residue is reduced as compared with a naturally occurring fibroin. It can be said that the modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with naturally occurring fibroin.
  • the modified fibroin according to the first embodiment has at least a domain sequence selected from GGX and GPGXX in REP (where X represents an amino acid residue other than glycine) as compared to naturally occurring fibroin.
  • One motif sequence may have an amino acid sequence corresponding to the substitution of one glycine residue in at least one or a plurality of the motif sequences with another amino acid residue.
  • the ratio of the motif sequence in which the above-described glycine residue is substituted with another amino acid residue may be 10% or more with respect to the entire motif sequence.
  • the modified fibroin according to the first embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and (A) n motif located on the most C-terminal side from the domain sequence.
  • Z is the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the domain sequence to the C-terminal of the domain sequence.
  • z / w is It may have an amino acid sequence that is 30% or more, 40% or more, 50% or more, or 50.9% or more.
  • the modified fibroin according to the first embodiment is obtained by substituting one glycine residue of the GGX motif with another amino acid residue. It is preferable that the content ratio of the amino acid sequence consisting of XGX is increased.
  • 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, and preferably 10% or less. More preferably, it is still more preferably 6% or less, still more preferably 4% or less, and particularly preferably 2% or less.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.
  • an amino acid sequence consisting of XGX is extracted from all REPs included in the sequence excluding the sequence from the domain motif (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • z / w (%) can be calculated by dividing z by w.
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, and further preferably 58.7% or more. 70% or more is even more preferable, and 80% or more is even more preferable. Although there is no restriction
  • the modified fibroin according to the first embodiment for example, from the cloned gene sequence of naturally-occurring fibroin, at least a part of the base sequence encoding a glycine residue is substituted to encode another amino acid residue. It can be obtained by modifying. At this time, one glycine residue in GGX motif and GPGXX motif may be selected as a glycine residue to be modified, or substitution may be performed so that z / w is 50.9% or more.
  • an amino acid sequence satisfying the above-described aspect can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • 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 other amino acid residue is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( M) hydrophobic amino acid residues such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and glutamic acid (E) residues are preferred, and valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residue is more preferable, and glutamine (Q) residue is more preferable.
  • modified fibroin As more specific examples of the modified fibroin according to the first embodiment, (1-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (1-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more of sequence identity with the amino acid sequence shown in No. 3, SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12.
  • the modified fibroin (1-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin.
  • the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence represented by SEQ ID NO: 3, in which every two (A) n motifs are deleted from the N-terminal side to the C-terminal side, and further before the C-terminal sequence.
  • One [(A) n motif-REP] is inserted into the.
  • the amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4.
  • the amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
  • the value of z / w in the amino acid sequence represented by SEQ ID NO: 1 is 46.8%.
  • the z / w values in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 12 are 58.7%, 70.1%, 66.1% and 70.0%.
  • the value of x / y at the ratio of the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 (described later) 1: 1.8 to 11.3 is: 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.
  • the modified fibroin (1-i) may consist of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin (1-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin of (1-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (1-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules.
  • affinity tag include a histidine tag (His tag).
  • His tag is a short peptide with about 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography) Can be used.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 5 (amino acid sequence containing a His tag).
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • an “epitope tag” using 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.
  • HA peptide sequence of hemagglutinin of influenza virus
  • myc tag peptide sequence of hemagglutinin of influenza virus
  • FLAG tag peptide sequence of hemagglutinin of influenza virus
  • a tag sequence that can be separated with 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 examples include (1-iii) the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (1-iv) SEQ ID NO: 8 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively.
  • amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
  • the modified fibroin (1-iii) may be composed of the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin (1-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin of (1-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (1-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the modified fibroin according to the second embodiment has an amino acid sequence in which the domain sequence has a reduced content of (A) n motif compared to naturally occurring fibroin. It can be said that the domain sequence of the modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) n motifs as compared to naturally occurring fibroin.
  • the modified fibroin according to the second embodiment may have an amino acid sequence corresponding to 10% to 40% deletion of the (A) n motif from naturally occurring fibroin.
  • the modified fibroin according to the second embodiment has a domain sequence of 1 to 3 (A) n motifs at least from the N-terminal side to the C-terminal side as compared with naturally-occurring fibroin ( A) It may have an amino acid sequence corresponding to deletion of the n motif.
  • the modified fibroin according to the second embodiment has a domain sequence that is at least two consecutive from the N-terminal side to the C-terminal side compared to naturally-occurring fibroin (A) deletion of the n motif, and It may have an amino acid sequence corresponding to the deletion of one (A) n motif repeated in this order.
  • the modified fibroin according to the second embodiment has an amino acid sequence corresponding to the deletion of every two (A) n motifs at least every two domain sequences from the N-terminal side to the C-terminal side. There may be.
  • the modified fibroin according to the second embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [[( A) The number of amino acid residues in the REP of the n motif-REP] unit is sequentially compared, and when the number of amino acid residues in the REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is The maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units that are 1.8 to 11.3 is x, and the total number of amino acid residues in the domain sequence is When y, x / y may have an amino acid sequence of 20% or more, 30% or more, 40% or more, or 50% or more.
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin.
  • the domain sequence is from the N-terminal side (left side): (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called n motif.
  • FIG. 1 includes 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 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison), pattern 4 (first REP and Comparison of the second REP).
  • pattern 1 compare 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 4th REP and 5th REP
  • pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison
  • pattern 4 first REP and Comparison of the second REP
  • the number of amino acid residues of each REP in the two adjacent [(A) n motif-REP] units selected is compared.
  • each pattern the number of all amino acid residues of two adjacent [(A) n motif-REP] units indicated by solid lines is added (not only REP but also (A) the number of amino acid residues of the n motif. is there.). Then, the total value added is compared, and the total value (maximum value of the total value) of the pattern having the maximum total value is set 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 of the domain sequence.
  • x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, and 70% or more. Is more preferably 75% or more, still more preferably 80% or more.
  • x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, and 70% or more. Is more preferably 75% or more, still more preferably 80% or more.
  • limiting in particular in the upper limit of x / y For example, you may be 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 / 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.
  • x / y is 46.4% Preferably, it is 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, It is especially preferable that it is 80% or more.
  • x / y is 46.4%
  • it is 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, It is especially preferable that it is 80% or more.
  • x / y in naturally derived fibroin will be described.
  • 663 types of fibroin (of which 415 types were derived from spiders) were extracted.
  • x / y is calculated from the amino acid sequence of naturally derived fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above-described calculation method.
  • FIG. 3 shows the results when the jagged ratio is 1: 1.9 to 4.1.
  • the horizontal axis indicates x / y (%), and the vertical axis indicates frequency.
  • x / y in naturally derived fibroin is less than 64.2% (the highest, 64.14%).
  • the modified fibroin according to the second embodiment includes, for example, one or a plurality of sequences encoding the n motif so that x / y is 64.2% or more from the cloned gene sequence of natural fibroin (A) Can be obtained by deleting.
  • an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to this may be modified.
  • modified fibroin As a more specific example of the modified fibroin according to the second embodiment, (2-i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in No. 2, SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12.
  • the modified fibroin (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 2 has the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin deleted from the N-terminal side to the C-terminal side every two (A) n motifs Furthermore, one [(A) n motif-REP] is inserted in front of the C-terminal sequence.
  • the amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting all GGX in REP of the amino acid sequence shown in SEQ ID NO: 2 with GQX.
  • the amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4.
  • the amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
  • the value of x / y of the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally-occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 4 is 93.4%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 12 is 89.8%.
  • the z / w values in the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 are 46.8%, 56.2%, 70.1% and 66. respectively. 1% and 80.4%.
  • the modified fibroin (2-i) may be composed of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and from the N-terminal side to the C-terminal side
  • 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 64.2% or more, where x is the maximum total value of the total number of bases and y is the total number of amino acid residues in the domain sequence.
  • the above-described modified fibroin may contain the above-described tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin containing the tag sequence examples include (2-iii) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 7 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively.
  • amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
  • the modified fibroin (2-iii) may be composed of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (2-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and from the N-terminal side to the C-terminal side.
  • the other X is the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3.
  • x / y is preferably 64.2% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the domain sequence has a reduced content of glycine residues in addition to the reduced content of the (A) n motif compared to the naturally derived fibroin.
  • the domain sequence of the modified fibroin is different from that of naturally occurring fibroin in addition to at least one or more glycine residues in REP. It can be said to have an amino acid sequence corresponding to substitution with an amino acid residue. That is, the modified fibroin having the characteristics of the modified fibroin according to the first embodiment described above and the modified fibroin according to the second embodiment. Specific aspects and the like are as described in the modified fibroin according to the first and second embodiments.
  • modified fibroin More specific examples of the modified fibroin according to the third embodiment include (3-i) an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, (3-ii) SEQ ID NO: 4, SEQ ID NO: Examples thereof include modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 10 or SEQ ID NO: 12. Specific embodiments of the modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.
  • the modified fibroin according to the fourth embodiment has a domain sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues having a large hydrophobicity index as compared with naturally occurring fibroin, And / or having an amino acid sequence that locally includes a region having a large hydrophobicity index corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index in REP.
  • the region where the hydrophobic index is locally large is preferably composed of 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). More preferably, it is a residue.
  • the modified fibroin according to the fourth embodiment has one or more amino acid residues in REP substituted with amino acid residues having a large hydrophobicity index and / or in REP compared to naturally occurring fibroin.
  • substitution, deletion or insertion of one or more amino acid residues as compared with naturally occurring fibroin And / or there may be amino acid sequence modifications corresponding to the addition.
  • the modified fibroin according to the fourth embodiment includes, for example, one or more hydrophilic amino acid residues (for example, amino acid residues whose hydrophobicity index is negative) in REP from the cloned gene sequence of naturally-occurring fibroin. It can be obtained by substituting with a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP.
  • hydrophilic amino acid residues for example, amino acid residues whose hydrophobicity index is negative
  • 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 It can also be obtained by designing an amino acid sequence corresponding to insertion of, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in 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 acids in 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 modified fibroin according to the fourth embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side (A) from the n motif of the domain sequence.
  • the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence obtained by removing the sequence up to the C-terminal from the domain sequence.
  • P, and (A) where the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is q / Q may have an amino acid sequence of 6.2% or more.
  • hydrophobicity index of amino acid residues As for the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of bio.p. 7”. 105-132). Specifically, the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
  • a sequence obtained by removing the sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m to the most C-terminal side from the domain (A) n motif to the C terminus of the domain sequence. (Hereinafter referred to as “array A”).
  • array A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated.
  • the average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in the four consecutive amino acid residues by 4 (number of amino acid residues).
  • 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). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of hydrophobicity index is 2.6 or more”, 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.
  • the total number of amino acid residues contained in sequence A is q.
  • the average value of the hydrophobicity index of four consecutive amino acid residues is 2
  • p / q is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, and 20% or more. It is still more preferable that it is 30% or more.
  • the upper limit of p / q is not particularly limited, but may be 45% or less, for example.
  • the modified fibroin according to the fourth embodiment includes, for example, one or a plurality of hydrophilic amino acid residues in REP (for example, an amino acid sequence of a cloned naturally-derived fibroin so as to satisfy the above p / q condition) Substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or one or more hydrophobic amino acids during REP By inserting a residue, it can be obtained by locally modifying the amino acid sequence including a region having a large hydrophobicity index.
  • an amino acid sequence satisfying the above p / q conditions can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index and / or one or more amino acid residues in REP.
  • modifications corresponding to 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 ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
  • modified fibroin As another specific example of the modified fibroin according to the fourth embodiment, (4-i) the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, or (4-ii) SEQ ID NO: 15, Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.
  • the modified fibroin (4-i) will be described.
  • the amino acid sequence shown in SEQ ID NO: 14 is an amino acid sequence in which alanine residues in the (A) n motif of (A) naturally derived fibroin are deleted so that the number of consecutive alanine residues is five.
  • the amino acid sequence shown in SEQ ID NO: 15 is inserted into the amino acid sequence shown in SEQ ID NO: 14 by two amino acid sequences (VLI) each consisting of 3 amino acid residues for every other REP, and shown in SEQ ID NO: 14. A part of amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence is almost the same.
  • the amino acid sequence shown in SEQ ID NO: 16 is obtained by inserting two alanine residues on the C-terminal side of each (A) n motif with respect to the amino acid sequence shown in SEQ ID NO: 14, and further, some glutamine (Q) residues. A group is substituted with a serine (S) residue, and a part of amino acids on the C-terminal side is deleted so as to be approximately the same as the molecular weight of the amino acid sequence represented by SEQ ID NO: 14.
  • the amino acid sequence represented by SEQ ID NO: 17 is obtained by inserting one amino acid sequence (VLI) consisting of 3 amino acid residues every other REP to the amino acid sequence represented by SEQ ID NO: 16.
  • the amino acid sequence shown in SEQ ID NO: 18 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues into the amino acid sequence shown in SEQ ID NO: 16 every other REP.
  • the modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18.
  • the modified fibroin (4-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18.
  • the modified fibroin of (4-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (4-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, and is located at the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the above-mentioned modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin containing a tag sequence (4-iii) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (4-iv) SEQ ID NO: 19, SEQ ID NO: 20 or A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21 can be mentioned.
  • amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 are the amino acid sequences represented by SEQ ID NO: 5 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18, respectively (His tag). Including a sequence and a hinge sequence).
  • the modified fibroin 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 (4-iv) includes 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 (4-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (4-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the modified fibroin according to the present embodiment includes, 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. It 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 a method such as amplification by polymerase chain reaction (PCR), cloning, modification by genetic engineering techniques, or chemical synthesis. can do.
  • the method for chemically synthesizing nucleic acids is not particularly limited. For example, based on the amino acid sequence information of fibroin obtained from the NCBI web database, etc., AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) A gene can be chemically synthesized by a method of linking oligonucleotides that are synthesized automatically by PCR or the like.
  • a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N terminus of the above amino acid sequence is synthesized. May be.
  • Regulatory sequences are sequences that control the expression of modified fibroin in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected depending on the type of host.
  • an inducible promoter that functions in the host cell and can induce expression of the modified fibroin may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), 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 a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like.
  • a vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like.
  • microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • a prokaryotic host is used as a vector for introducing a nucleic acid encoding a modified fibroin
  • a prokaryotic host for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like can be mentioned.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeast include yeasts belonging to the genus 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 a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC 37115) and YEp24 (ATCC 37051).
  • a method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • electroporation 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, etc. .
  • 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 a host in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. 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.
  • Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism.
  • Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc.
  • Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source examples include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, 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 cells and digested products thereof can be used.
  • inorganic salts for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and 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 the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • Isolation and purification of the expressed modified fibroin can be performed by a commonly used method.
  • the host cells are recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a manton. A host cell is crushed with a Gaurin homogenizer, dynomill, etc., and a cell-free extract is obtained.
  • a method usually used for isolation and purification of modified fibroin that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method by DE, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), or a resin such as S-Sepharose FF (manufactured by Pharmacia) Electrophoresis such as cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Use methods such as law alone or in combination It is possible to obtain a purified product.
  • a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA
  • the modified fibroin when expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to collect the modified fibroin insoluble substance as a precipitate fraction.
  • the recovered insoluble form of modified fibroin can be solubilized with a protein denaturant.
  • a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.
  • the modified fibroin when secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture with a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the artificial fibroin fiber according to the present embodiment can be produced by a known spinning method. That is, for example, when producing an artificial fibroin fiber containing a modified fibroin as a main component, first, the modified fibroin produced according to the above-described method is converted into dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), Alternatively, it is added to a solvent such as hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator and dissolved to prepare a dope solution. Next, the target artificial fibroin fiber can be obtained by spinning using this dope solution by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning. Preferred spinning methods include wet spinning or dry wet spinning.
  • FIG. 5 is an explanatory view schematically showing an example of a spinning device for producing artificial fibroin fibers.
  • a spinning device 10 shown in FIG. 5 is an example of a spinning device for dry and wet spinning, and includes an extrusion device 1, an undrawn yarn production device 2, a wet heat drawing device 3, and a drying device 4.
  • the dope solution 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8.
  • the dope solution may be filled into a cylinder and extruded 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, the modified fibroin is coagulated, and a fibrous coagulated body is formed.
  • the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn.
  • 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 solidified body is supplied to the drying device 4 and dried in the yarn path 22, and an artificial fibroin fiber is obtained as the wound thread body 5.
  • Reference numerals 18a to 18g denote thread guides.
  • the coagulation liquid 11 may be any solution 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 appropriately contain water.
  • the temperature of the coagulation liquid 11 is preferably 0 to 30 ° C.
  • the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and 1.4 to 4.0 ml / hour More preferably it is time.
  • the distance through which the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) has only to be a length that allows efficient desolvation, for example, 200 to 500 mm. It is.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min.
  • the residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes.
  • stretching pre-stretching
  • the coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as necessary.
  • the stretching performed when obtaining the artificial fibroin fiber for example, dry stretching is also adopted in addition to the pre-stretching performed in the coagulating liquid tank 20 and the wet heat stretching performed in the stretching bath 21.
  • Wet and hot stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, and in steam heating.
  • the temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C.
  • undrawn yarn or predrawn yarn
  • Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C.
  • an undrawn yarn or predrawn yarn
  • an undrawn yarn can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching.
  • wet heat stretching and dry heat stretching can be appropriately combined.
  • the final draw ratio of the lower limit of the undrawn yarn (or predrawn yarn) is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times. Above, 7 times or more, 8 times or more, 9 times or more, and upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less 12 times or less, 11 times or less, and 10 times or less.
  • the artificial fibroin fiber according to this embodiment is obtained by spinning the above-described modified fibroin and contains the above-described modified fibroin as a main component.
  • the artificial fibroin fiber according to this embodiment preferably has a shrinkage rate defined by the following formula exceeding 7%. Thereby, it can shrink
  • Shrinkage rate ⁇ 1 ⁇ (length of artificial fibroin fiber subjected to shrinkage processing including contact with water having less than boiling point / length of artificial fibroin fiber before being subjected to shrinkage processing) ⁇ ⁇ 100 (%)
  • the shrinkage rate of the artificial fibroin fiber according to this embodiment is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, and further preferably 40% or more. More preferably, it is 48% or more, still more preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more.
  • the upper limit of shrinkage is usually 80% or less.
  • Shrinkage processing includes contacting artificial fibroin fiber with water having a boiling point (hereinafter also referred to as “contacting step”).
  • the shrinking process may further include drying the artificial fibroin fiber after contact with water (after the contact step) (hereinafter also referred to as “drying step”).
  • the temperature of the water to be brought into contact with the artificial fibroin fiber in the contact step may be less than the boiling point. Thereby, the handleability, the workability of shrinkage processing, and the like are improved. Further, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit value of the water temperature is preferably 10 ° C or higher, more preferably 40 ° C or higher, and further preferably 70 ° C or higher. . The upper limit of the water temperature is preferably 90 ° C. or lower.
  • the method for bringing water into contact with the artificial fibroin fiber is not particularly limited.
  • a method of immersing the artificial fibroin fiber in water a method of spraying water on the artificial fibroin fiber at room temperature or in a heated steam state, and the high humidity where the artificial fibroin fiber is filled with water vapor Examples include exposure to the environment.
  • the method of immersing the artificial fibroin fiber in water is preferable in the contact step because the shrinkage time can be effectively shortened and the processing equipment can be simplified.
  • the contact step when the artificial fibroin fiber is brought into contact with water in a relaxed state, the artificial fibroin fiber may not only contract but also shrink like a wave.
  • the contact step is performed in a state where the artificial fibroin fiber is not relaxed, for example, the artificial fibroin fiber is brought into contact with water below the boiling point while being tensioned (pulled) in the fiber axis direction. May be.
  • the drying step is a step of drying the artificial fibroin fiber that has undergone the contact step. Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known drying equipment of contact type or non-contact type can be used.
  • the drying temperature is not limited as long as it is lower than the temperature at which, for example, the protein contained in the artificial fibroin fiber is decomposed or the artificial fibroin fiber is thermally damaged.
  • the temperature is in the range of 20 to 150 ° C., and the temperature is preferably in the range of 50 to 100 ° C. When the temperature is in this range, the artificial fibroin fiber is dried more quickly and efficiently without thermal damage of the artificial fibroin fiber or degradation of the protein contained in the artificial fibroin fiber.
  • the drying time is appropriately set according to the drying temperature or the like. For example, a time during which the influence on the quality and physical properties of the artificial fibroin fiber due to overdrying can be eliminated as much as possible is adopted.
  • the artificial fibroin fiber according to the present invention can be highly contracted, for example, the highly contracted artificial fibroin fiber can be obtained through the above-described shrinking process (contacting step and drying step as necessary).
  • the high-shrinkage artificial fibroin fiber according to this embodiment has a shrinkage rate defined as described above exceeding 7%.
  • the shrinkage ratio of the high shrinkage artificial fibroin fiber is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, and still more preferably 40% or more, More preferably, it is 48% or more, particularly preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more.
  • the upper limit of shrinkage is usually 80% or less.
  • FIG. 6 is an explanatory view schematically showing an example of an apparatus for shrinking an artificial fibroin fiber.
  • the apparatus 40 shown in FIG. 6 includes a feed roller 42 that feeds out artificial fibroin fibers, a winder 44 that winds up the highly shrinkable artificial fibroin fibers 38, a water bath 46 that performs a contact step, and a dryer 48 that performs a drying step. , And is configured.
  • the feed roller 42 can be attached with a wound product of the artificial fibroin fiber 36, and the artificial fibroin fiber 36 is continuously formed from the wound product of the artificial fibroin fiber 36 by rotation of an electric motor (not shown). Can be delivered automatically and automatically.
  • the winder 44 can continuously and automatically wind up the high shrinkage artificial fibroin fiber 38 produced through the contact step and the drying step by the rotation of an electric motor (not shown). ing.
  • the feeding speed of the artificial fibroin fiber 36 by the feed roller 42 and the winding speed of the highly shrinkable artificial fibroin fiber 38 by the winder 44 can be controlled independently of each other.
  • the water bath 46 and the dryer 48 are arranged side by side between the feed roller 42 and the winder 44 on the upstream side and the downstream side in the feeding direction of the artificial fibroin fiber 36.
  • the apparatus 40 shown in FIG. 6 has relay rollers 50 and 52 that relay the artificial fibroin fiber 36 that travels from the feed roller 42 toward the winder 44.
  • the water bath 46 has a heater 54, and hot water 47 heated by the heater 54 is accommodated in the water bath 46.
  • a tension roller 56 is installed in the water bath 46 so as to be immersed in the hot water 47.
  • the artificial fibroin fiber 36 fed out from the feed roller 42 travels toward the winder 44 while being immersed in the hot water 47 while being wound around the tension roller 56 in the water bath 46. It has become.
  • the immersion time of the artificial fibroin fiber 36 in the hot water 47 is appropriately controlled according to the traveling speed of the artificial fibroin fiber 36.
  • the dryer 48 has a pair of hot rollers 58.
  • the pair of hot rollers 58 can be wound around the artificial fibroin fiber 36 that moves away from the water bath 46 and travels toward the winder 44 side.
  • the artificial fibroin fiber 36 immersed in the hot water 47 in the water bath 46 is heated by the pair of hot rollers 58 in the dryer 48 and dried, and then sent out further toward the winder 44. It has become.
  • the apparatus 40 When manufacturing the high-shrinkage artificial fibroin fiber 38 using the apparatus 40 having such a structure, first, for example, winding of the artificial fibroin fiber 36 spun using the spinning apparatus 10 shown in FIG. The circulation is mounted on the feed roller 42. Next, the artificial fibroin fiber 36 is continuously fed from the feed roller 42 and immersed in hot water 47 in the water bath 46. At this time, for example, the winding speed of the winder 44 is set slower than the feeding speed of the feed roller 42. Thereby, since the artificial fibroin fiber 36 is contracted by contact with the hot water 47 in a state where the artificial fibroin fiber 36 is tensioned so as not to relax between the feed roller 42 and the winder 44, occurrence of crimping can be prevented.
  • the artificial fibroin fiber 36 contracted in the hot water 47 in the water bath 46 is heated by a pair of hot rollers 58 of the dryer 48.
  • the contracted artificial fibroin fiber 36 is dried to obtain a highly contracted artificial fibroin fiber 38.
  • the artificial fibroin fiber 36 can be further contracted, and the length can be kept unchanged.
  • the obtained high-shrinkage artificial fibroin fiber 38 is wound up by a winder 44 to obtain a wound product of the high-shrinkage artificial fibroin fiber 38.
  • the drying means is constituted by the dry heat plate 64.
  • the intended high-shrinkage artificial fibroin fiber 38 can be manufactured automatically and continuously and extremely easily.
  • FIG. 7 is an explanatory view schematically showing another example of an apparatus for shrinking artificial fibroin fibers.
  • FIG. 7A shows a processing apparatus that performs the contact step provided in the apparatus
  • FIG. 7B shows a drying apparatus that performs the drying step provided in the apparatus.
  • the apparatus shown in FIG. 7 includes a processing device 60 that performs a contact step with respect to the artificial fibroin fiber 36, and a drying device 62 that dries the artificial fibroin fiber 36 subjected to the contact step with the processing device 60.
  • a processing device 60 that performs a contact step with respect to the artificial fibroin fiber 36
  • a drying device 62 that dries the artificial fibroin fiber 36 subjected to the contact step with the processing device 60.
  • the processing device 60 shown in FIG. 7A omits the dryer 48 from the device 40 shown in FIG. 6, and replaces the feed roller 42, the water bath 46, and the winder 44 with the artificial fibroin.
  • the fiber 36 has a structure in which the fibers 36 are arranged in order from the upstream side to the downstream side in the traveling direction.
  • the artificial fibroin fiber 36 fed from the feed roller 42 is immersed in hot water 47 in the water bath 46 and contracted before being wound up by the winder 44. .
  • the artificial fibroin fiber 36 contracted in the hot water 47 is configured to be wound up by the winder 44.
  • the 7B includes a feed roller 42 and a winder 44, and a dry heat plate 64.
  • the dry heat plate 64 is disposed between the feed roller 42 and the winder 44 so that the dry heat surface 66 contacts the artificial fibroin fiber 36 and extends along the traveling direction.
  • the artificial fibroin fiber 36 can be further shrunk by controlling the ratio of the feed speed of the feed roller 42 and the winding speed of the winder 44, for example. It is also possible not to change the length.
  • the artificial fibroin fiber 36 is shrunk by the processing device 60, and then the artificial fibroin fiber 36 is dried by the drying device 62.
  • High shrinkage man-made fibroin fiber 38 can be produced.
  • the processing apparatus 60 may be configured with only the water bath 46 by omitting the feed roller 42 and the winder 44 from the processing apparatus 60 shown in FIG.
  • high-shrinkage artificial fibroin fibers are manufactured in a so-called batch system.
  • Modified fibroin having the amino acid sequence represented by SEQ ID NO: 7 (PRT399), modified fibroin having the amino acid sequence represented by SEQ ID NO: 8 (PRT380), modified fibroin having the amino acid sequence represented by SEQ ID NO: 9 (PRT410), SEQ ID NO: A modified fibroin (PRT799) having the amino acid sequence shown by 13 was designed.
  • the amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin, wherein (A) n motif is deleted every two from the N-terminal side to the C-terminal side. Further, an amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminal to the amino acid sequence in which one [(A) n motif-REP] is inserted before the C-terminal sequence. .
  • amino acid sequence represented by SEQ ID NO: 8 is represented by SEQ ID NO: 5 at the N-terminal with respect to the amino acid sequence in which all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin are replaced with GQX. Added amino acid sequence (including His tag).
  • the amino acid sequence represented by SEQ ID NO: 9 is the amino acid sequence represented by SEQ ID NO: 5 at the N-terminus (with His tag) compared to the amino acid sequence in which all GGXs in REP of the amino acid sequence represented by SEQ ID NO: 2 are replaced with GQX Included).
  • the amino acid sequence shown in SEQ ID NO: 13 is a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted).
  • the amino acid sequence represented by SEQ ID NO: 5 (including the His tag) is added to the N-terminus of the amino acid sequence in which the His tag is added to the C-terminus of the sequence obtained by repeating 4 times.
  • the nucleic acids encoding the four types of designed modified fibroin were synthesized respectively.
  • the nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon.
  • These four types of nucleic acids were cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • 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 (Table 2) containing ampicillin so that the OD 600 was 0.005.
  • the culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
  • the seed culture solution was added to a jar fermenter to which 500 ml of production medium (Table 3 below) was added so that the OD 600 was 0.05.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration.
  • 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target modified fibroin.
  • the culture solution was centrifuged, and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of a band of a size corresponding to the target modified fibroin depending on the addition of IPTG Expression was confirmed.
  • the washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 60 ° C. And stirred for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). White aggregated protein obtained after dialysis was collected by centrifugation. Water was removed from the recovered aggregated protein with a freeze dryer to obtain the desired modified fibroin freeze-dried powder.
  • 8M guanidine buffer 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • spun and drawn artificial fibroin fibers were produced by dry and wet spinning using a spinning device similar to the spinning device 10 shown in FIG.
  • the spinning device used is a spinning device 10 shown in FIG. 5, and a second undrawn yarn producing device (a first bath) and a wet heat drawing device 3 (third bath) between the undrawn yarn producing device 2 (first bath) and the second undrawn yarn producing device (3rd bath) A second bath).
  • the dry and wet spinning conditions are as follows. Extrusion nozzle diameter: 0.2 mm Liquid and temperature in the first to third baths: see Table 4 Total draw ratio: see Table 4 Drying temperature: 60 ° C.
  • a plurality of artificial fibroin fibers for testing each having a length of 30 cm were cut out from the wound product of artificial fibroin fibers obtained in Production Examples 1 to 19.
  • the plurality of artificial fibroin fibers were bundled to obtain an artificial fibroin fiber bundle having a fineness of 150 denier.
  • 0.8 g of lead weight was attached to each artificial fibroin fiber bundle, and in this state, each artificial fibroin fiber bundle was immersed in water having a temperature shown in Tables 5 to 8 for 10 minutes (shrinkage processing 1). Thereafter, the length of each artificial fibroin fiber bundle was measured in water.
  • the artificial fibroin fiber according to the present invention has a sufficiently high shrinkage rate.

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Abstract

L'invention concerne des fibres de fibroïne synthétique qui contiennent une fibroïne modifiée, et qui présentent un coefficient de retrait défini par la formule suivante supérieur à 7%. coefficient de retrait =（longueur des fibres de fibroïne synthétique après exécution d'un processus de retrait incluant une mise en contact avec une eau de température inférieure au point d'ébullition / longueur des fibres de fibroïne synthétique avant exécution du processus de retrait）｝×１００（％）
PCT/JP2018/008800 2017-03-10 2018-03-07 Fibres de fibroïne synthétique WO2018164190A1 (fr)

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WO2020067545A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Fil composite et son procédé de fabrication, et tissu
WO2020067553A1 (fr) * 2018-09-28 2020-04-02 株式会社島精機製作所 Procédé de fabrication de fil filé de protéine
WO2020067546A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Fil composite, son procédé de production et tissu
WO2020067513A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Coton en fibre synthétique
WO2020067547A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Fibres de fibroïne modifiée
WO2020145363A1 (fr) * 2019-01-09 2020-07-16 Ｓｐｉｂｅｒ株式会社 Fibroïne modifiée
WO2020175702A1 (fr) * 2019-02-28 2020-09-03 株式会社ゴールドウイン Tissu étanche perméable à l'humidité et son procédé de production
WO2020262602A1 (fr) * 2019-06-28 2020-12-30 Ｓｐｉｂｅｒ株式会社 Cuir synthétique, procédé de fabrication associé et agent adhésif pour cuir synthétique
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EP3476859A4 (fr) * 2016-06-23 2019-12-11 Spiber Inc. Fibroïne modifiée
JPWO2020067553A1 (ja) * 2018-09-28 2021-09-02 株式会社島精機製作所 タンパク質紡績糸の製造方法
JP2022024192A (ja) * 2018-09-28 2022-02-09 Ｓｐｉｂｅｒ株式会社 複合糸、及びその製造方法、並びに布地
CN112714813A (zh) * 2018-09-28 2021-04-27 丝芭博株式会社 改性丝心蛋白纤维
JP7446578B2 (ja) 2018-09-28 2024-03-11 Ｓｐｉｂｅｒ株式会社 人造繊維綿
WO2020067547A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Fibres de fibroïne modifiée
JP2022024194A (ja) * 2018-09-28 2022-02-09 Ｓｐｉｂｅｒ株式会社 複合糸、及びその製造方法、並びに布地
WO2020067553A1 (fr) * 2018-09-28 2020-04-02 株式会社島精機製作所 Procédé de fabrication de fil filé de protéine
JP2022024191A (ja) * 2018-09-28 2022-02-09 Ｓｐｉｂｅｒ株式会社 人造繊維綿
JP7466872B2 (ja) 2018-09-28 2024-04-15 株式会社島精機製作所 タンパク質紡績糸の製造方法
WO2020067546A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Fil composite, son procédé de production et tissu
WO2020067513A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Coton en fibre synthétique
WO2020067545A1 (fr) * 2018-09-28 2020-04-02 Ｓｐｉｂｅｒ株式会社 Fil composite et son procédé de fabrication, et tissu
WO2020145363A1 (fr) * 2019-01-09 2020-07-16 Ｓｐｉｂｅｒ株式会社 Fibroïne modifiée
EP3910096A4 (fr) * 2019-01-09 2023-05-24 Spiber Inc. Fibroïne modifiée
CN113260745A (zh) * 2019-01-09 2021-08-13 丝芭博株式会社 改造丝心蛋白
WO2020175702A1 (fr) * 2019-02-28 2020-09-03 株式会社ゴールドウイン Tissu étanche perméable à l'humidité et son procédé de production
WO2020262602A1 (fr) * 2019-06-28 2020-12-30 Ｓｐｉｂｅｒ株式会社 Cuir synthétique, procédé de fabrication associé et agent adhésif pour cuir synthétique
WO2021002437A1 (fr) * 2019-07-03 2021-01-07 Ｓｐｉｂｅｒ株式会社 Complexe de fibroïne modifiée et procédé pour la production de celui-ci

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