WO2019066053A1 - Procédé et dispositif de production de fibres protéiques, procédé de traitement de fibres protéiques - Google Patents

Procédé et dispositif de production de fibres protéiques, procédé de traitement de fibres protéiques Download PDF

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WO2019066053A1
WO2019066053A1 PCT/JP2018/036532 JP2018036532W WO2019066053A1 WO 2019066053 A1 WO2019066053 A1 WO 2019066053A1 JP 2018036532 W JP2018036532 W JP 2018036532W WO 2019066053 A1 WO2019066053 A1 WO 2019066053A1
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protein
amino acid
seq
fiber
raw material
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PCT/JP2018/036532
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Japanese (ja)
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秀人 石井
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Ｓｐｉｂｅｒ株式会社
小島プレス工業株式会社
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Priority to JP2019545187A priority Critical patent/JPWO2019066053A1/ja
Publication of WO2019066053A1 publication Critical patent/WO2019066053A1/fr

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    • 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
    • D—TEXTILES; PAPER
    • D02—YARNS; MECHANICAL FINISHING OF YARNS OR ROPES; WARPING OR BEAMING
    • D02J—FINISHING OR DRESSING OF FILAMENTS, YARNS, THREADS, CORDS, ROPES OR THE LIKE
    • D02J1/00—Modifying the structure or properties resulting from a particular structure; Modifying, retaining, or restoring the physical form or cross-sectional shape, e.g. by use of dies or squeeze rollers
    • D02J1/22—Stretching or tensioning, shrinking or relaxing, e.g. by use of overfeed and underfeed apparatus, or preventing stretch

Definitions

  • the present disclosure relates to a method of producing protein fiber, a device for producing protein fiber, and a method of processing protein fiber.
  • various protein fibers have been produced using spinning methods such as dry spinning, wet spinning, and dry-wet spinning.
  • spinning methods such as dry spinning, wet spinning, and dry-wet spinning.
  • a target protein fiber is obtained by extruding a spinning solution linearly in air or a predetermined coagulating solution and coagulating.
  • a drawing process of drawing the protein fiber is performed (see, for example, Patent Document 1).
  • the present disclosure describes a method for producing a protein fiber and a device for producing a protein fiber, which can easily produce a protein fiber having high tensile strength and high elongation.
  • the present disclosure also describes a method of processing protein fibers that can impart high tensile strength and high elongation to protein fibers by simple processing on protein fibers.
  • the method for producing a protein fiber comprises a stretching step of stretching a protein raw material fiber containing protein, a heating step of heating the protein raw fiber after the drawing step, and a heating step simultaneously or after the heating step And a relaxation and contraction step which is performed and which relaxes and shrinks the protein raw material fiber in a heated state by the heating step.
  • high tensile strength and high elongation can be achieved by relaxing (without tension or pulling) the protein raw material fiber which has been heated through the stretching step and contracting it.
  • the protein fiber which it has can be manufactured simply.
  • the drawing step may include a drawing ratio adjustment step of adjusting the drawing ratio of the protein raw material fiber.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • the at least one of the relaxation and contraction step and the heating step may include a contraction amount adjustment step of adjusting the contraction amount of the protein raw material fiber.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • the contraction amount adjustment step at least one of the relaxation amount of the protein raw material fiber in the relaxation and contraction step and the heating temperature of the protein raw material fiber in the heating step may be adjusted.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • the contraction amount adjustment step is included in the relaxation contraction step, and in the relaxation contraction step, the protein material fiber is continuously delivered at a predetermined delivery rate and continuously wound at a take-up rate slower than the delivery rate,
  • the protein raw material fiber may be relaxed and contracted, and the amount of relaxation of the protein raw material fiber may be controlled by adjusting at least one of the delivery speed and the winding speed in the contraction amount adjustment step. In this case, the productivity can be improved by continuous production of protein fibers.
  • the heating temperature of the protein raw material fiber in the heating step may be less than 180 ° C. In this case, a drop in tensile strength due to heating can be advantageously prevented.
  • the protein may be a structural protein.
  • a structural protein fiber having high tensile strength and high elongation can be easily produced.
  • the structural protein may be fibroin.
  • fibroin fibers having high tensile strength and high elongation can be easily produced.
  • the fibroin may be spider silk fibroin.
  • spider silk fibroin fibers having high tensile strength and high elongation can be easily produced.
  • An apparatus for producing a protein fiber comprises a spinning means for spinning a protein raw fiber containing protein, a drawing means for drawing a protein raw fiber spun by the spinning means, and a drawing means.
  • a protein fiber having high tensile strength and high elongation can be simply manufactured by relaxing and shrinking the protein raw material fiber in a heated state after the drawing process. it can.
  • the drawing means may have a drawing ratio adjusting means for adjusting the drawing ratio of the protein raw material fiber.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • the relaxation and contraction means may have a relaxation amount adjusting means for adjusting the amount of relaxation of the protein raw material fiber.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • the relaxation and contraction means continuously takes up the delivery means for continuously delivering the protein raw fiber at a predetermined delivery rate, and continuously takes up the protein raw fiber delivered by the delivery means at a take-up speed slower than the delivery rate
  • the slack amount adjusting means may comprise a speed adjusting means for adjusting at least one of the delivery speed of the delivery means and the winding speed of the winding means. In this case, the productivity can be improved by continuous production of protein fibers.
  • the apparatus for producing protein fibers may further comprise temperature control means for controlling the heating temperature of the protein raw material fibers in the heating means.
  • temperature control means for controlling the heating temperature of the protein raw material fibers in the heating means.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • the method for processing protein fiber comprises a drawing step of drawing protein fiber containing protein, a heating step of heating the protein fiber subjected to the drawing step, and a heating step simultaneously or after the heating step. And a relaxation and contraction step which is carried out and which relaxes and shrinks the protein fiber in the heated state by the heating step.
  • the protein fiber processing method by stretching and shrinking the protein fiber in a heated state through the drawing process, the protein fiber can be easily processed to have high tensile strength and high elongation. Can be given.
  • the drawing step may include a drawing ratio adjustment step of adjusting the drawing ratio of the protein fiber.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • the relaxation / contraction step and / or the heating step may include a contraction amount adjustment step of adjusting the contraction amount of the protein fiber.
  • the tensile strength and elongation of the protein fiber can be arbitrarily controlled.
  • protein fibers having high tensile strength and high elongation can be conveniently produced.
  • FIG. 1 is a schematic view showing the domain sequence of modified fibroin.
  • FIG. 2 is a diagram showing the distribution of the value of z / w (%) of naturally occurring fibroin.
  • FIG. 3 shows the distribution of x / y (%) values of naturally occurring fibroin.
  • FIG. 4 is a schematic view showing an example of the domain sequence of the modified fibroin.
  • FIG. 5 is a schematic view showing an example of the domain sequence of the modified fibroin.
  • FIG. 6 is a schematic view of a protein fiber manufacturing apparatus according to an embodiment of the present disclosure.
  • FIG. 7 is a figure which shows the speed control means and temperature control means which can be provided in the high temperature heating furnace in FIG.
  • the method for producing a protein fiber according to the present embodiment is carried out simultaneously with or after the heating step of heating the protein raw material fiber that has undergone the drawing step, the heating step of heating the protein raw material fiber that has undergone the drawing step, and the heating step. And a relaxation and contraction step for relaxing and contracting the protein raw material fiber in a heated state by the heating step.
  • the apparatus for producing protein fiber according to the present embodiment includes a spinning means for spinning protein raw material fiber containing protein, a stretching means for stretching protein raw material fiber spun by the spinning means, and a protein raw material stretched by the stretching means A heating means for heating the fiber, and a relaxation and contraction means for relaxing and shrinking the protein raw material fiber in a heated state by the heating means are provided.
  • protein A protein fiber produced according to the production method of the present invention, or a protein raw material fiber as a raw material, contains, as a main component, a protein that gives a fiber that shrinks upon contact with moisture.
  • the protein is not particularly limited, and may be one produced by a microorganism or the like by genetic recombination technology, or may be one produced synthetically, or one obtained by purifying a protein of natural origin It may be Also, the protein may be, for example, a structural protein.
  • the structural protein refers to a protein that forms a biological structure or a protein derived therefrom. That is, the structural protein may be a naturally occurring structural protein, and is a modified protein in which a portion (for example, 10% or less of the amino acid sequence) of the amino acid sequence is altered based on the amino acid sequence of the naturally occurring structural protein. It may be
  • structural proteins include fibroin, collagen, resilin, elastin and keratin, and proteins derived therefrom, and the like.
  • the fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin.
  • the structural protein may be silk fibroin, spider silk fibroin or a combination thereof. When silk fibroin and spider silk fibroin are used in combination, the proportion of silk fibroin may be, for example, 40 parts by mass or less, 30 parts by mass or less, or 10 parts by mass or less with respect to 100 parts by mass of spider silk fibroin.
  • the modified fibroin according to this 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 that contains.
  • 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.
  • An N-terminal sequence and a C-terminal sequence are typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.
  • modified fibroin means artificially produced fibroin (artificial fibroin).
  • the modified fibroin may be fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or fibroin whose amino acid sequence is identical to that of naturally occurring fibroin.
  • naturally-derived fibroin 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 the domain sequence
  • the “modified fibroin” may be one obtained by directly using the amino acid sequence of naturally occurring fibroin, or one obtained by modifying the amino acid sequence based on the amino acid sequence of naturally occurring fibroin (eg, cloned natural origin)
  • the amino acid sequence may be modified by modifying the gene sequence of fibroin), or artificially designed and synthesized without relying on naturally occurring fibroin (eg, a nucleic acid encoding the designed amino acid sequence) It may be one having a desired amino acid sequence by chemical synthesis).
  • domain sequence refers to a crystal region specific to fibroin (typically corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically the REP of the amino acid sequence).
  • Amino acid sequence which corresponds to the following formula 1: [(A) n motif -REP] m , or formula 2: [(A) n motif -REP] m- (A) n motif Means sequence.
  • (A) n motif indicates an amino acid sequence mainly comprising an alanine residue, 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 (A) n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning it consists only of alanine residues).
  • At least seven of the (A) n motifs present in the domain sequence may consist of only alanine residues.
  • REP represents an amino acid sequence composed of 2 to 200 amino acid residues.
  • the REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • m is an integer of 2 to 300, and may be an integer of 10 to 300.
  • the plurality of (A) n motifs may be identical to each other or different from each other.
  • the plurality of REPs may be identical amino acid sequences to each other or different amino acid sequences.
  • the modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned naturally occurring fibroin gene sequence.
  • Can be obtained by modifying Substitutions, deletions, insertions and / or additions of amino acid residues can be carried out by methods 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 occurring fibroin is a protein comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif -REP] m- (A) n Motif Specifically, for example, fibroin produced by insects or spiders can be mentioned.
  • fibroin produced by insects include Bombyx mori (Bombyx mori), Quwaco (Bombyx mandarina), pemphigus (Antheraea yamamai), moth (Anteraea pernyi), moth (Eriogyna pyretorum), moth (Pilosamia Cynthia ricini) ), Silk proteins produced by silkworms such as silkworms (Samia cynthia), chestnut beetles (Caligura japonica), tussah silkworms (Antheraea mylitta), and muga silkworms (Antheraea assama), and larvae of the hornets (Vespa simillima xanthoptera) Hornet silk protein is mentioned.
  • insect-produced fibroin include, for example, silkworm fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • the fibroins produced by the spiders include, for example, spiders belonging to the genus Araneus such as spider spiders, spider spiders, spider spiders, blue spider spiders, and spider spiders, spider spiders (genus Neoscona) such as spider spiders, spider spiders, spider spiders and spiders , Spiders belonging to the genus Pronus (Pronus), such as Torino Fundamas, spiders belonging to the genus Torino Fundama (Cyrtarachne) such as Torino Fundamas, and Otorino Fundames, such as spiders such as Togegumo and Tibusegumo Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Gasteracantha and those belonging to the genus Ordgarius A spider belonging to the genus Angiope (Argiope), a spider belonging to the genus Angiope, a spider
  • spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC 47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 derived from Nephila clavipes (genbank accession number AAC 04504 (amino acid sequence) ), U37520 (base sequence)), major ampullate spidro n 1 [Latrodectus hesperus derived] (GenBank accession No.
  • ABR68856 amino acid sequence
  • EF 595246 base sequence
  • dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession No. AAL 32 472 (amino acid sequence), AF 441 245 (base sequence ), Major ampullate spidroin 1 [from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ 973 155 (base sequence)), and major ampullate spidroin 2 [Euprosthenops australi (GenBank Accession No. CAM 32249.
  • Naturally derived fibroin further include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” are described as keywords among sequences including INV as DIVISION among sequence information registered in NCBI GenBank.
  • the sequence can be confirmed by extracting a specified product string from CDS, and a described sequence of a specific string from SOURCE to TISSUE TYPE.
  • the modified fibroin according to this embodiment may be a modified silk (silk) fibroin (a modified amino acid sequence of a silk protein produced by silkworm), or a modified spider silk fibroin (a spider silk protein produced by spiders)
  • the amino acid sequence may be modified).
  • modified spider silk fibroin is preferred.
  • a modified fibroin (first modified fibroin) derived from the large nasogastric silkworm silk protein produced in the large vein of the spider, a domain sequence with a reduced content of glycine residues (A) a modified fibroin (a third modified fibroin) having a domain sequence with a reduced content of n motif, a content of a glycine residue, and (A) n Modified fibroin (fourth modified fibroin) having a reduced content of motif, modified fibroin having a domain sequence including a region locally having a large hydrophobicity index (fifth modified fibroin), and content of glutamine residue And modified fibroin (sixth modified fibroin) having a reduced domain sequence.
  • the first modified fibroin includes a protein comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m .
  • the amino acid residue number of the (A) n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, and an integer of 10 to 20 Is still more preferred, the integer of 4 to 16 is even more preferred, the integer of 8 to 16 is particularly preferred, and the integer of 10 to 16 is most preferred.
  • the number of amino acid residues constituting the REP is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues More preferably, it is 20 to 75 residues.
  • the total number of residues of glycine, serine and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) n motif-REP] m is an amino acid residue
  • the total number is preferably 40% or more, more preferably 60% or more, and still more preferably 70% or more.
  • the first modified fibroin comprises a unit of the amino acid sequence represented by the formula 1: [(A) n motif-REP] m , and the amino acid sequence whose C-terminal sequence is shown in any one of SEQ ID NOs: 1 to 3 or It may be a polypeptide which is an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.
  • the amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of 50 C-terminal amino acids 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 identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 has 29 residues removed from the C terminus of the amino acid sequence shown in SEQ ID NO. It is identical to the amino acid sequence.
  • the amino acid sequence represented by (1-i) SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: Mention may be made of modified fibroins which comprise amino acid sequences with% or more sequence identity.
  • the sequence identity is preferably 95% or more.
  • the amino acid sequence shown by SEQ ID NO: 4 is the first amino acid sequence of the amino acid sequence of ADF3 to which an amino acid sequence (SEQ ID NO: 5) consisting of an initiation codon, His10 tag and HRV3C protease (Human rhinovirus 3C protease) recognition site is added at the N terminus.
  • the 13th repeat region is about doubled and the translation is mutated to terminate at amino acid residue 1154.
  • the amino acid sequence at the C-terminus of the amino acid sequence shown in SEQ ID NO: 4 is identical to the amino acid sequence shown in SEQ ID NO: 3.
  • the modified fibroin of (1-i) may consist of the amino acid sequence shown by SEQ ID NO: 4.
  • the second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin.
  • the second modified fibroin can be said to have an amino acid sequence corresponding to the replacement of at least one glycine residue in REP with another amino acid residue as compared to naturally occurring fibroin .
  • GGX and GPGXX in REP (wherein G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine) in the second modified fibroin in comparison with the naturally derived fibroin in its domain sequence In which at least one glycine residue in at least one or more motif sequences is substituted with another amino acid residue.
  • the percentage of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more with respect to the entire motif sequence.
  • the second modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and from the above domain sequence to the most C-terminally located (A) n motif from the above domain sequence
  • the alanine residue number relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more It is more preferred that there be 100%, meaning that it consists only of alanine residues.
  • the second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by replacing 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, still more preferably 10% or less, and 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 calculation method of the content ratio (z / w) of the amino acid sequence consisting of XGX described below.
  • fibroin modified fibroin or naturally-derived fibroin
  • fibroin containing a domain sequence represented by the formula 1: [(A) n motif-REP] m , (A) n located most C-terminally from the domain sequence
  • An amino acid sequence consisting of XGX is extracted from all the REP contained in the sequence excluding the sequence from the motif to the C-terminus 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-terminus to the C-terminus 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 fibroin derived from spiders) were extracted.
  • z / w was calculated by the above-mentioned calculation method. The results are shown in FIG.
  • the horizontal axis of FIG. 2 indicates z / w (%) and the vertical axis indicates frequency.
  • z / w in all naturally occurring fibroin is less than 50.9% (highest, 50.86%).
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, and 70% or more It is further more preferred that the ratio is 80% or more.
  • the upper limit of z / w is not particularly limited, and may be, for example, 95% or less.
  • the second modified fibroin can be obtained, for example, by replacing at least a part of the nucleotide sequence encoding a glycine residue from the cloned gene sequence of naturally occurring fibroin to encode another amino acid residue You can get it.
  • a glycine residue to be modified one glycine residue in the GGX motif and the GPGXX motif may be selected, or z / w may be substituted so as to be 50.9% or more.
  • it can be obtained by designing an amino acid sequence satisfying the above embodiment from the amino acid sequence of naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence may be modified corresponding to the insertion and / or addition.
  • the above 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 residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ), Hydrophilic amino acid residues such as lysine (K) residue and glutamic acid (E) residue are preferable, and valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine ( F) The residue and glutamine (Q) residue are more preferred, and glutamine (Q) residue is even more preferred.
  • SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT 525) or SEQ ID NO: 9 (Met)
  • the modified fibroin of (2-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 6 is one in which all GGX in the REP of the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin is replaced with GQX.
  • the amino acid sequence shown by SEQ ID NO: 7 is such that every other (A) n motif is deleted from the amino acid sequence shown by SEQ ID NO. [(A) n Motif-REP] is inserted into.
  • the amino acid sequence shown by SEQ ID NO: 8 inserts two alanine residues at the C-terminal side of each (A) n motif of the amino acid sequence shown by SEQ ID NO: 7, and further contains some glutamine (Q) residues.
  • SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 7 (however, several amino acid residues at the C-terminal side of the region are substituted).
  • a predetermined hinge sequence and a His tag sequence are added to the C terminus of the sequence repeated four times.
  • the value of z / w in the amino acid sequence shown in SEQ ID NO: 10 is 46.8%.
  • the value of x / y in the Giza ratio (described later) 1: 1.8 to 11.3 of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
  • the modified fibroin of (2-i) may consist of the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown 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 comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and However, when X represents the total number of amino acid residues of the amino acid sequence consisting of amino acid residues other than glycine) as z, and the total number of amino acid residues of REP in the above domain sequence as w, z / w Is preferably 50.9% or more.
  • the second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This makes it possible to isolate, immobilize, detect, visualize, etc., the modified fibroin.
  • an affinity tag utilizing specific affinity (binding, affinity) with another molecule can be mentioned.
  • a histidine tag (His tag) can be mentioned as a specific example of an affinity tag.
  • the His tag is a short peptide consisting of 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel, so isolation of the modified fibroin by metalating metal chromatography It can be used to Specific examples of the tag sequence include, for example, the amino acid sequence shown in SEQ ID NO: 11 (His tag sequence and amino acid sequence including 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
  • epitope tags utilizing antigen-antibody reactions can also be used.
  • a peptide (epitope) showing antigenicity as a tag sequence an antibody against the epitope can be bound.
  • the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like.
  • tag sequence can be separated by a specific protease
  • modified fibroin from which the tag sequence has been separated can also be recovered by subjecting the protein adsorbed via the tag sequence to a protease treatment.
  • modified fibroin containing a tag sequence (2-iii) SEQ ID NO: 12 (PRT 380), SEQ ID NO: 13 (PRT 410), SEQ ID NO: 14 (PRT 525) or SEQ ID NO: 15 (PRT 799)
  • a modified fibroin can be mentioned, which comprises an amino acid sequence having 90% or more sequence identity with the sequence or (2-iv) the amino acid sequence shown in 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 (PRT 313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are respectively SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9
  • amino acid sequence shown in SEQ ID NO: 11 (including His tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence shown.
  • the modified fibroin of (2-iii) may consist of the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and However, when X represents the total number of amino acid residues of the amino acid sequence consisting of amino acid residues other than glycine) as z, and the total number of amino acid residues of REP in the above domain sequence as w, z / w Is preferably 50.9% or more.
  • the second modified fibroin may comprise a secretion signal for releasing the protein produced in the recombinant protein production system outside the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the third modified fibroin has an amino acid sequence in which the content of the (A) n motif is reduced as compared to naturally occurring fibroin.
  • the domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or more (A) n motifs as compared to naturally occurring fibroin.
  • the third modified fibroin may have an amino acid sequence corresponding to 10-40% of the (A) n motif deleted 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 deletion of two consecutive (A) n motifs whose domain sequences are at least N-terminal to C-terminal as compared to naturally occurring fibroin, and one (A The amino acid sequence may correspond to the fact that the deletion of the n motif is repeated in this order.
  • the third modified fibroin may have an amino acid sequence corresponding to the deletion of (A) n motif every other two domain sequences from at least 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 toward the C terminal side
  • the ratio of the number of amino acid residues of the other REP is 1.8 to Assuming that the maximum value of the sum of the amino acid residue numbers of two adjacent [(A) n motif-REP] units to be 11.3 is x and the total amino acid residue number of the domain sequence is y
  • it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more.
  • the alanine residue number relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more It is more preferred that there be 100%, meaning that it consists only of alanine residues.
  • FIG. 1 shows domain sequences obtained by removing the N- and C-terminal sequences from the modified fibroin. From the N-terminal side (left side), the domain sequence is (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called n motif.
  • the number of amino acid residues of each REP in two adjacent selected [(A) n motif-REP] units is compared.
  • the comparison is carried out by determining the ratio of the number of amino acid residues of the other, assuming that the smaller number of amino acid residues is 1.
  • each pattern the numbers of all amino acid residues of two adjacent [(A) n motif-REP] units shown by the solid line are added (not only REP, but also the number of amino acid residues of (A) n motif is there.). Then, the summed total values are compared, and the total value (maximum value of the total values) of the patterns for which the total value is the largest is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the largest.
  • 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, still more preferably 65% or more, still more preferably 70% or more It is more preferably 75% or more, still more preferably 80% or more.
  • x / y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, still more preferably 70% or more It is more preferably 75% or more, still more preferably 80% or more.
  • the upper limit of x / y may be, for example, 100% or less.
  • x / y is preferably 89.6% or more, and in the case of a Giza ratio of 1: 1.8 to 3.4, x It is preferable that / y is 77.1% or more, and when the Giza ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the Giza 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% or more Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, and 80% or more. Being particularly preferred.
  • 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.
  • x / y in naturally derived fibroin is less than 64.2% in all cases (highest, 64.14%).
  • the third modified fibroin deletes one or more of the sequences encoding the (A) n motif so that x / y is 64.2% or more from the cloned naturally-occurring fibroin gene sequence It can be obtained by Also, for example, from the amino acid sequence of naturally occurring fibroin, an amino acid sequence corresponding to deletion of one or more (A) n motifs so that x / y is 64.2% or more is designed and designed It can also be obtained by chemically synthesizing a nucleic acid encoding the above amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to the above may be modified.
  • the third modified fibroin (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT 525) or SEQ ID NO: 9 (Met)
  • the modified fibroin of (3-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 17 is different from the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin from every N terminal side toward C terminal side (A) n
  • the motif is deleted, and one [(A) n motif-REP] is inserted in front of the C-terminal sequence.
  • the amino acid sequence shown by 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 Giza ratio 1: 1.8 to 11.3 of the amino acid sequence (corresponding to naturally occurring fibroin) represented by SEQ ID NO: 10 is 15.0%.
  • the amino acid sequence shown by SEQ ID NO: 17 and the value of x / y in the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%.
  • the value of x / y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%.
  • the value of x / y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%.
  • the values of z / w in the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
  • the modified fibroin of (3-i) may consist of the amino acid sequence shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (3-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown 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 comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from N-terminal to C-terminal Sequentially comparing the number of amino acid residues of REP of two [(A) n motif-REP] units adjacent to each other, and assuming that the number of amino acid residues of REP having a small number of amino acid residues is 1, Amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues of REP is 1.8 to 11.3 (the Giza ratio is 1: 1.8 to 11.3) It is preferable that x / y be 64.2% or more, where x is the maximum value of the sum total of the number of bases and x is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may contain the above-described tag sequence at either or both of the N-terminus and the C-terminus.
  • modified fibroin containing the tag sequence 3-iii) SEQ ID NO: 18 (PRT 399), SEQ ID NO: 13 (PRT 410), SEQ ID NO: 14 (PRT 525) or SEQ ID NO: 15 (PRT 799)
  • a modified fibroin can be mentioned, which comprises an amino acid sequence having 90% or more sequence identity with the sequence or (3-iv) the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 .
  • amino acid sequences shown by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 correspond to SEQ ID NO: 11 at the N-terminus of the amino acid sequences shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • the amino acid sequence (including His tag sequence and hinge sequence) is added.
  • the modified fibroin of (3-iii) may consist of the amino acid sequence shown by 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 shown by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and N-terminal to C-terminal Sequentially comparing the number of amino acid residues of REP of two [(A) n motif-REP] units adjacent to each other, and assuming that the number of amino acid residues of REP having a small number of amino acid residues is 1, The maximum value of the sum of the amino acid residue numbers of two adjacent [(A) n motif-REP] units in which the ratio of the amino acid residue number of REP is 1.8 to 11.3 is x.
  • x / y is 64.2% or more, where y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may contain a 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 fourth modified fibroin has an amino acid sequence in which the content of the glycine residue is reduced in addition to the content of the (A) n motif being reduced as compared to the naturally derived fibroin of the domain sequence. It is possessed.
  • the domain sequence of the fourth modified fibroin has at least one or more glycine residues in the REP in addition to the deletion of at least one or more (A) n motifs as compared to naturally occurring fibroin It can be said to have an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the second modified fibroin described above and the third modified fibroin. Specific embodiments and the like are as described in the second modified fibroin and the third modified fibroin.
  • modified fibroin As more specific examples of 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) Or the amino acid sequence shown in SEQ ID NO: 14 (PRT 525) or SEQ ID NO: 15 (PRT 799), 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 fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Specific embodiments of the modified fibroin comprising the amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 9, 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 is that its domain sequence has one or more amino acid residues in REP replaced with an amino acid residue having a large hydrophobicity index, as compared to naturally occurring fibroin, and / or REP It may have an amino acid sequence including a region having a locally large hydrophobicity index corresponding to insertion of one or more hydrophobicity index large amino acid residues therein.
  • the region locally having a large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
  • the amino acid residue having a large hydrophobicity index mentioned above is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) It is more preferable that it is a residue.
  • one or more amino acid residues in the REP are replaced with an amino acid residue having a large hydrophobicity index, as compared with naturally occurring fibroin, and / or one or more in the REP.
  • substitution, deletion, insertion and / or addition of one or more amino acid residues as compared with naturally occurring fibroin There may be amino acid sequence modifications corresponding to those described above.
  • the fifth modified fibroin is, for example, a hydrophobic amino acid residue remaining in one or more hydrophilic amino acid residues (for example, an amino acid residue having a negative hydrophobicity index) in REP from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting a group (for example, an amino acid residue whose hydrophobicity index is plus) and / or inserting one or more hydrophobic amino acid residues into the REP. Also, for example, from the amino acid sequence of naturally-derived fibroin, one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues, and / or one or more hydrophobic amino acid residues in REP.
  • an amino acid sequence corresponding to the insertion of X can also be obtained by designing an amino acid sequence corresponding to the insertion of X, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in the REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally derived 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 , and from the (A) n motif located most at the C-terminal end to the C-terminus of the domain sequence
  • Let p be the total number of amino acid residues contained in a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the above sequence from the above domain sequence,
  • p / q is 6
  • hydrophobicity index of amino acid residues
  • known indices Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. Use 105-132).
  • the hydrophobicity index (hydropathy index, hereinafter also referred to as "HI" of each amino acid is as shown in Table 1 below.
  • sequence A [(A) n motif-REP] m to the sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence (Hereinafter, referred to as "sequence A") is used.
  • sequence A the average value of the hydrophobicity index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the sum of HI of each amino acid residue contained in 4 consecutive amino acid residues by 4 (the number of amino acid residues).
  • the average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used to calculate an average of 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is identified. Even if a certain amino acid residue corresponds to "a series of 4 amino acid residues in which the average value of the hydrophobicity index is 2.6 or more", the region is included as one amino acid residue become. And, the total number of amino acid residues contained in the region is p. In addition, 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, still more preferably 10% or more, and preferably 20% or more. Still more preferably, it is 30% or more.
  • the upper limit of p / q is not particularly limited, and may be, for example, 45% or less.
  • the fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP such that the amino acid sequence of the cloned naturally-derived fibroin satisfies the above p / q condition.
  • a hydrophobic amino acid residue eg, an amino acid residue with a positive hydrophobicity index
  • insertion of one or more hydrophobic amino acid residues into the REP By doing this, it can be obtained by locally modifying the amino acid sequence including the region having a large hydrophobicity index.
  • one or more amino acid residues in the REP are replaced with an amino acid residue having a large hydrophobicity index, and / or one or more amino acids in the REP as compared to naturally occurring fibroin, and / or
  • modification 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, and 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.
  • the fifth modified fibroin (5-i) an amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), Or (5-ii) a modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 19 consists of three amino acid residues for every REP, except for the domain sequence at the C-terminal end of the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410)
  • the amino acid sequence (VLI) is inserted in two places, and a part of glutamine (Q) residues is replaced with a serine (S) residue and a part of amino acids at the C-terminal side is deleted.
  • the amino acid sequence shown by SEQ ID NO: 20 is one obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues for every REP in addition to the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525). is there.
  • the amino acid sequence shown by SEQ ID NO: 21 is one obtained by inserting two amino acid sequences (VLI) consisting of three amino acid residues for every REP, to the amino acid sequence shown by SEQ ID NO: 8.
  • the modified fibroin of (5-i) may consist of the amino acid sequence shown 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 a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and is most C-terminally located (A) n Amino acids included in a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence Assuming that the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence from the domain sequence is q. And 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 the tag sequence examples include (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) A modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24).
  • amino acid sequences shown by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences shown by SEQ ID NO: 11 (His tag) at the N terminus of the amino acid sequences shown by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 respectively (Including sequence and hinge sequence).
  • the modified fibroin of (5-iii) may consist of the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) comprises an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 and is most C-terminally located (A) n Amino acids included in a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence Assuming that the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence from the domain sequence is q. And p / q is preferably 6.2% or more.
  • the fifth 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 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 GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more.
  • the upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
  • GPGXX motif content is a value calculated by the following method.
  • Formula 1 [(A) n Motif -REP] m
  • Formula 2 [(A) n Motif -REP] m- (A) fibroin containing a domain sequence represented by n motif (modified fibroin or naturally derived In fibroin)
  • the number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the (A) n motif located most C-terminal to the C-terminus of the domain sequence from the domain sequence Let s be the number obtained by multiplying the total number by 3 (that is, the total number of G and P in the GPGXX motif) be s, and the sequence from the (A) n motif located closest to the C terminal to the C terminal of the domain sequence GPGXX motif content ratio is calculated as s / t, where t is the total number of amino acid residues of all REP excluding (A) n motif
  • GPGXX motif content “the sequence obtained by removing the sequence from the (A) n motif located at the most C terminal side to the C terminus of the domain sequence from the domain sequence” is “most C terminal side (A)
  • a sequence from the n motif to the C terminus of the domain sequence (sequence corresponding to REP) may contain a sequence with low correlation with the sequence characteristic of fibroin, and m is small If this is the case (that is, if the domain sequence is short), this affects the result of calculation of the GPGXX motif content, so this effect is eliminated.
  • GPGXX motif is located at the C-terminal of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.
  • FIG. 5 is a schematic view showing the domain sequence of modified fibroin.
  • all the REPs are "the sequence from the (A) n motif located at the most C-terminal end to the C-terminal end of the domain sequence removed from the domain sequence" (the sequence shown in "region A” in FIG.
  • the sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%. .
  • glucose 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), the sequence from the (A) n motif located closest to the C terminus to the C terminus of the domain sequence is all removed from the domain sequence (sequence corresponding to "region A" in Fig. 5).
  • the total number of glutamine residues contained in the area as u, except from the most located C-terminal side (a) sequence domain sequence from n motif to the C-terminal domain sequence, further (a) n
  • the glutamine residue content is calculated as u / t, where t is the total number of amino acid residues of all REPs excluding the motif.
  • the reason why “a sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence” is the reason described above It is similar.
  • the sixth modified fibroin corresponds to deletion of one or more glutamine residues in the REP or substitution of another amino acid residue 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 a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue.
  • the hydrophobicity index of amino acid residues is as shown in Table 1.
  • amino acid residues having a larger hydrophobicity index than glutamine residues isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), 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.
  • the amino acid residue is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). More preferably, it is an amino acid residue selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
  • the sixth modified fibroin preferably has a hydrophobicity of -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, and 0.3 or more. It is further more preferable that the ratio be 0.4 or more, and 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-
  • A) fibroin containing a domain sequence represented by n motif modified fibroin or naturally derived In fibroin
  • the sequence from the (A) n motif located closest to the C terminus to the C terminus of the domain sequence is all removed from the domain sequence (sequence corresponding to "region A" in Fig. 5).
  • the total of the hydrophobicity index of each amino acid residue in the region is v
  • the sequence from the (A) n motif located most C-terminal to the C-terminus of the domain sequence is removed from the domain sequence
  • the hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REP excluding n motif.
  • the reason for targeting “a sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence is excluded from the domain sequence” in the calculation of the hydrophobicity of REP is the reason described above and It is similar.
  • the sixth modified fibroin has its domain sequence deleted one or more glutamine residues in the REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in the REP
  • the modification corresponding to substitution of the amino acid with another amino acid residue there may be a modification of the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues.
  • the sixth modified fibroin may, for example, delete one or more glutamine residues in the REP from the cloned naturally occurring fibroin gene sequence and / or one or more glutamine residues in the REP It can be obtained by substitution of amino acid residues of Also, for example, one or more glutamine residues in REP are deleted from the amino acid sequence of naturally-derived fibroin, and / or one or more glutamine residues in REP are replaced with another amino acid residue. Particularly, it can be obtained by designing a corresponding amino acid sequence 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 Modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 29 (Met-PRT 918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT 698) or SEQ ID NO: 32 (Met-PRT966), or (6-ii) 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 31 or SEQ ID NO: 32 Mention may be made of modified fibroin which comprises the amino acid sequence it has.
  • the modified fibroin of (6-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 25 is one in which all QQs in the amino acid sequence (Met-PRT410) shown by SEQ ID NO: 7 are replaced with VL.
  • the amino acid sequence shown by SEQ ID NO: 26 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with TS, and the remaining Q is replaced with A.
  • the amino acid sequence shown by SEQ ID NO: 27 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VL, and the remaining Q is replaced with I.
  • the amino acid sequence shown by SEQ ID NO: 28 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VI, and the remaining Q is replaced with L.
  • the amino acid sequence shown by SEQ ID NO: 29 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VF, and the remaining Q is replaced with I.
  • the amino acid sequence shown by SEQ ID NO: 30 is one in which all QQs in the amino acid sequence (Met-PRT 525) shown by SEQ ID NO: 8 are replaced with VL.
  • the amino acid sequence shown by SEQ ID NO: 31 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 8 are replaced with VL, and the remaining Q is replaced with I.
  • amino acid sequence shown by SEQ ID NO: 32 is the same as the one shown in SEQ ID NO: 7 (Met-PRT410), in which the QQ in the double repeated sequence of the region of 20 domain sequences is replaced with VF, And the remaining Q is replaced by I.
  • amino acid sequences shown by 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 and SEQ ID NO: 32 all have glutamine residue content of 9% or less Yes (Table 2).
  • the modified fibroin of (6-i) consists of the amino acid sequence shown by 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 or SEQ ID NO: 32 It may be.
  • the modified fibroin of (6-ii) has 90% or more amino acid sequence shown by 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 or SEQ ID NO: 32 Containing an amino acid sequence having the sequence identity of
  • the modified fibroin of (6-ii) is also a domain 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 a sequence.
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Moreover, it is preferable that the modified fibroin of (6-ii) 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 makes it possible to isolate, immobilize, detect, visualize, etc., the modified fibroin.
  • modified fibroin containing the tag sequence are (6-iii) SEQ ID NO: 33 (PRT 888), SEQ ID NO: 34 (PRT 965), SEQ ID NO: 35 (PRT 889), SEQ ID NO: 36 (PRT 916), SEQ ID NO: 37 (PRT 918), SEQ ID NO: 38 (PRT 699), SEQ ID NO: 39 (PRT 698) or modified fibroin comprising the amino acid sequence shown by SEQ ID NO: 40 (PRT 966), or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 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 SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 are SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 respectively.
  • SEQ ID NO: 28 SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32
  • the amino acid sequence shown in SEQ ID NO: 11 was added to the N terminus of the amino acid sequence shown in It is a thing.
  • amino acid sequence shown by SEQ ID NO: 40 has a glutamine residue content of 9% or less (Table 3).
  • the modified fibroin of (6-iii) consists of the amino acid sequence 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 or SEQ ID NO: 40 It may be.
  • the modified fibroin of (6-iv) has 90% or more of the amino acid sequence represented 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 or SEQ ID NO: 40 Containing an amino acid sequence having the sequence identity of
  • the modified fibroin of (6-iv) is also a domain 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 a sequence.
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. Moreover, it is preferable that the modified fibroin of (6-iv) has a GPGXX motif content of 10% or more.
  • the sixth 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 is at least two or more of the characteristics possessed by the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin It may be a modified fibroin having the characteristics of
  • a protein comprising a domain sequence represented by Formula 2: [REP2] p (wherein, in Formula 2, p represents an integer of 5 to 300.
  • REP2 is Gly-XY)
  • X and Y each represent any amino acid residue other than Gly, and a plurality of REP2 may be identical amino acid sequences to each other or may be different amino acid sequences).
  • SEQ ID NO: 41 a protein comprising the amino acid sequence shown by SEQ ID NO: 41 can be mentioned.
  • the amino acid sequence shown by SEQ ID NO: 41 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (NCBI GenBank accession numbers: CAA56335.1, GI: 3702452) obtained from the NCBI database.
  • the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 11 is added to the N-terminus of the amino acid sequence from residue 301 to residue 540.
  • REP3 As a protein derived from resilin, for example, a protein comprising a domain sequence represented by the formula 3: [REP3] q (wherein, in the formula 3, q represents an integer of 4 to 300.
  • REP3 is a Ser-J-J- An amino acid sequence consisting of Tyr-Gly-U-Pro is shown, J is any amino acid residue, preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr, and U is any option.
  • the amino acid residue is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser.
  • the plurality of REP 4 may be identical to each other or different from each other. Can be mentioned.
  • the amino acid sequence shown by SEQ ID NO: 2 is the amino acid sequence of resilin (NCBI GenBank accession numbers NP 611 157, Gl: 24654243), wherein Thr at position 87 is substituted with Ser and Asn at position 95
  • the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 11 is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which
  • a protein derived from elastin for example, a protein having an amino acid sequence such as Accession Nos. AAC98395 (human), I47076 (sheep), NP786966 (bovine) of GenBank of NCBI can be mentioned.
  • a protein comprising the amino acid sequence shown in SEQ ID NO: 43 can be mentioned.
  • the amino acid sequence set forth in SEQ ID NO: 43 is the amino acid sequence set forth in SEQ ID NO: 11 at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of NCBI GenBank accession number AAC 98395 (tag sequence And hinge arrangement) are added.
  • a protein derived from keratin for example, type I keratin of Capra hircus etc. can be mentioned. Specifically, a protein comprising the amino acid sequence shown in SEQ ID NO: 44 (amino acid sequence of accession number ACY30466 of GenBank of NCBI) can be mentioned.
  • the structural protein described above and the protein derived from the structural protein can be used singly or in combination of two or more.
  • a protein fiber and a protein contained as a main component in a protein raw material fiber are transformed, for example, with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing the nucleic acid by the selected host.
  • the nucleic acid can be produced by a method of amplification and cloning by polymerase chain reaction (PCR) or the like, or a method of chemical synthesis, using a gene encoding a natural structural protein.
  • the method for chemically synthesizing nucleic acid is not particularly limited, and, for example, AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. based on the amino acid sequence information of structural proteins obtained from the NCBI web database etc.
  • the gene can be chemically synthesized by a method of ligating the oligonucleotide synthesized at step S by PCR or the like.
  • a nucleic acid encoding a protein consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N terminus of the above amino acid sequence is synthesized It is also good.
  • the regulatory sequence is a sequence that controls the expression of a recombinant protein in a host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host.
  • a promoter an inducible promoter which functions in a host cell and is capable of inducible expression of a target protein may be used.
  • An inducible promoter is a promoter that can control transcription due to the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as temperature, osmotic pressure or an increase or decrease in pH value.
  • the type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector and the like.
  • a vector capable of autonomous replication in a host cell or capable of integration into the host chromosome and containing a promoter at a position capable of transcribing a nucleic acid encoding a target protein is suitably used. .
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.
  • Preferred examples of the prokaryotic host include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like.
  • Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like.
  • microorganisms belonging to the genus Serratia include Serratia liquofaciens and the like.
  • Bacillus subtilis and the like can be mentioned.
  • microorganism belonging to the genus Microbacterium examples include, for example, Microbacterium ammoniafilum and the like.
  • microorganisms belonging to the genus Brevibacterium examples include Brevibacterium divaricatam and the like.
  • microorganisms belonging to the genus Corynebacterium examples include Corynebacterium ammoniagenes and the like.
  • Pseudomonas for example, Pseudomonas putida etc. can be mentioned.
  • examples of vectors for introducing a nucleic acid encoding a target protein include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Application Laid-Open No. 2002-238569) and the like can be mentioned.
  • Eukaryotic hosts can include, for example, yeast and filamentous fungi (molds and the like).
  • yeast the yeast which belongs to Saccharomyces genus, Pichia genus, Schizosaccharomyces genus etc. can be mentioned, for example.
  • filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
  • examples of vectors into which a nucleic acid encoding a target protein is introduced include YEP13 (ATCC 37115), YEp24 (ATCC 37051), and the like.
  • a method of introducing the expression vector into the host cell any method of introducing DNA into the host cell can be used. For example, a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], 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, secretion production, fusion protein expression and the like can be performed according to the method described in Molecular Cloning 2nd Edition, etc. .
  • a protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium.
  • the method of culturing the host in a culture medium can be carried out according to a method usually used for culturing the host.
  • the culture medium When the host is a prokaryote such as E. coli or a eukaryote such as yeast, the culture medium contains a carbon source which can be used by the host, a nitrogen source, inorganic salts and the like, and the medium can efficiently culture the host. If it is, either a natural culture medium or a synthetic culture medium may be used.
  • the carbon source may be any as long as the above-mentioned transformed microorganism can assimilate, for example, glucose, fructose, sucrose and molasses containing them, carbohydrates such as starch and starch hydrolysate, acetic acid and propionic acid etc. Organic acids and alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include, for example, ammonium, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • inorganic salts for example, potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • the culture of a prokaryote such as E. coli or a eukaryote such as yeast can be performed under aerobic conditions such as shake culture or submerged aeration culture, for example.
  • 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 culture is preferably maintained at 3.0 to 9.0. Adjustment of the pH of the culture medium can be carried out using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia and the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed.
  • an inducer may be added to the medium as needed.
  • indole acrylic An acid or the like may be added to the medium.
  • Isolation and purification of the expressed protein can be performed by a commonly used method. For example, when the protein is expressed in a dissolved state in cells, after completion of culture, host cells are recovered by centrifugation and suspended in an aqueous buffer, and then sonicator, French press, Manton Gaulin The host cells are disrupted by a homogenizer, dynomill or the like to obtain a cell-free extract.
  • resin such as diethylaminoethyl (DEAE) -s
  • the host cell When the protein is expressed in the form of an insoluble form in cells, the host cell is similarly recovered and then disrupted and centrifuged to recover the insoluble form of the protein as a precipitate fraction.
  • the insoluble matter of the recovered protein can be solubilized with a protein denaturant.
  • a purified preparation of protein can be obtained by the same isolation and purification method as described above.
  • the protein When the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture according to 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 protein raw material fiber is obtained by spinning the above-described protein, and contains the above-described protein as a main component.
  • the protein raw material fiber can be produced by a known spinning method. That is, for example, when producing a protein raw material fiber containing spider silk fibroin as a main component, first, spider silk fibroin produced according to the method described above is dimethylsulfoxide (DMSO), N, N-dimethylformamide (DMF) ) Or hexafluoroisopronol (HFIP), formic acid or the like to dissolve it to prepare a dope solution. At this time, inorganic salts may be added as necessary.
  • DMSO dimethylsulfoxide
  • DMF N, N-dimethylformamide
  • HFIP hexafluoroisopronol
  • the dope solution is used to carry out spinning by a known spinning method such as wet, dry or dry-wet method, thereby adding it together with an inorganic salt as a dissolution promoter and dissolving to prepare a dope solution. Then, using this dope solution, it can be spun by a known spinning method such as wet spinning, dry spinning or dry-wet spinning to obtain a target protein raw material fiber.
  • a known spinning method such as wet, dry or dry-wet method
  • FIG. 6 is a schematic view of a protein fiber manufacturing apparatus according to an embodiment of the present disclosure.
  • FIG. 7 is a figure which shows the speed control means and temperature control means which can be provided in the high temperature heating furnace in FIG.
  • the manufacturing apparatus 10 shown in FIG. 6 is an apparatus capable of easily manufacturing a protein fiber having high tensile strength and high elongation by spinning a protein raw material fiber and further subjecting the protein raw material fiber to a predetermined treatment. It is.
  • the manufacturing apparatus 10 includes a spinning device (spinning means) 25 for spinning the protein raw material fiber 36, and a high temperature heating relaxation device 40 for heating and shrinking the protein raw material fiber 36 spun by the spinning device 25 at a high temperature There is.
  • the spinning process, the drawing process, and the shrinking process by relaxation contraction in a heated state are continuously performed.
  • a protein fiber 50 having high tensile strength and high elongation can be produced with high productivity.
  • the spinning device 25 is, for example, a spinning device for dry-wet spinning, and includes an extrusion device 1, a coagulation device 2, a washing device 3, and a drying device 4 in this order from the upstream side.
  • the extrusion device 1 has a storage tank 7 in which a dope solution (spinning stock solution) 6 is stored.
  • the coagulation device 2 has a coagulation bath 20, in which coagulation liquid 11 (for example, methanol) is stored.
  • the dope solution 6 is pushed out from a nozzle 9 provided by opening a air gap 19 between the dope solution 6 and the coagulating solution 11 by a gear pump 8 attached to the lower end of the storage tank 7.
  • the extruded dope 6 is supplied into the coagulating liquid 11 through the air gap 19.
  • the solvent is removed from the dope solution 6 in the coagulation solution 11 to coagulate the protein.
  • the coagulating solution 11 may be any solution which can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone.
  • the coagulation liquid 11 may contain water as appropriate.
  • the temperature of the coagulating solution 11 is preferably 0 to 30 ° C.
  • the distance by which the coagulated protein passes through the coagulation liquid 11 is a length that can ensure the residence time of the protein raw material fiber 36 in the coagulation liquid 11 It is good if there is.
  • the residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, or may be 0.5 to 1 minute. Alternatively, stretching (pre-stretching) may be performed in the coagulating solution 11.
  • the cleaning device 3 has a cleaning bath 21, and the cleaning bath 12 stores cleaning liquid 12 (for example, water).
  • the proteins coagulated in the coagulation bath 20 are guided to the washing bath 21 and washed with the washing solution 12. This protein is sent to the drying device 4 by the first nip roller 13 and the second nip roller 14 installed in the washing bath 21.
  • the cleaning device 3 has the first nip roller 13 and the second nip roller 14 so as to have the function of a stretching device for stretching the protein raw material fiber 36. That is, the first nip roller 13 and the second nip roller 14 constitute a stretching means 26 for stretching the protein raw material fiber 36.
  • the rotational speed of the second nip roller 14 is set to be faster than the rotational speed of the first nip roller 13, whereby a protein raw material fiber 36 drawn at a magnification corresponding to the rotational speed ratio is obtained.
  • Reference numerals 18a to 18e denote yarn guides.
  • the first nip roller 13 and the second nip roller 14 may be provided with draw ratio adjusting means for adjusting the draw ratio of the protein raw material fiber 36 by adjusting the rotational speed of these.
  • the stretching unit 26 may have a controller that transmits a control signal to a drive unit that rotationally drives the first nip roller 13 and the second nip roller 14.
  • the stretching performed in the washing bath 21 may be so-called wet heat stretching performed in warm water, in a solution in which an organic solvent or the like is added to warm water, or the like.
  • the temperature of the wet heat drawing may be, for example, 0 to 90 ° C., preferably 20 to 70 ° C., and more preferably 30 to 60 ° C.
  • the draw ratio of the undrawn yarn (or pre-drawn yarn) in wet heat drawing may be, for example, 1 to 10 times or 2 to 8 times.
  • the lower limit of the final draw ratio is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times that of the undrawn yarn (or pre-drawn yarn).
  • the upper limit value is preferably 100 times or less, 80 times or less, 60 times or less, 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, 10 times or less.
  • cleaning apparatus 3 serves as an extending
  • stretching apparatus may each be provided separately.
  • a stretching device may be provided on the downstream side of the cleaning device 3 in the traveling direction of the protein raw material fiber 36.
  • the stretching device may be provided between the washing device 3 and the drying device 4.
  • a stretching device may be provided on the upstream side of the cleaning device 3.
  • the stretching device may be provided, for example, between the coagulation bath 20 and the cleaning device 3.
  • the protein raw material fiber 36 drawn in the washing liquid 12 is dried when passing through the drying device 4 after leaving the inside of the washing bath 21.
  • the drying device 4 includes, for example, a drying-type drying oven 17.
  • a delivery roller 31 and a winding roller 32 are provided.
  • the protein raw material fibers 36 stay in the drying furnace 17 for a predetermined staying time by the delivery roller 31 and the take-up roller 32, and are then sent to the high temperature heating and relaxing device 40.
  • the rotational speed of the take-up roller 32 may be set to be faster than the rotational speed of the delivery roller 31, so that the protein raw material fiber 36 may be drawn at a magnification corresponding to the rotational speed ratio.
  • a heater (not shown) is provided.
  • the temperature in the drying oven 17, that is, the drying temperature of the protein raw material fiber 36 is, for example, 80.degree.
  • An oiling device 30 may be provided between the cleaning device 3 and the drying device 4.
  • the high-temperature heat relaxation device 40 is provided on the downstream side of the spinning device 25 in the traveling direction of the protein raw material fiber 36.
  • the high-temperature heating and relaxing apparatus 40 which is a dry pressure-reduction apparatus, includes, for example, a high-temperature heating furnace 43 of a dry heat type.
  • a delivery roller (delivery means) 41 and a take-up roller (take-up means) 42 are provided in the high temperature heating furnace 43.
  • the delivery roller 41 and the take-up roller 42 both have a cylindrical shape, and the protein raw material fiber 36 is wound around the circumferential surface thereof.
  • the protein raw material fibers 36 stay in the high-temperature heating furnace 43 for a predetermined staying time by the delivery roller 41 and the take-up roller 42, and are then taken up by a winder.
  • the rotational speed of the take-up roller 42 is set to be slower than the rotational speed of the delivery roller 41, whereby the protein raw fiber 36 is relaxed at a magnification corresponding to the rotational speed ratio. That is, the delivery roller 41 is configured to continuously deliver the protein raw material fiber 36 at a predetermined delivery speed.
  • the take-up roller 42 is configured to communicably wind the protein raw material fiber 36 delivered by the delivery roller 41 at a take-up speed that is slower than the delivery speed of the delivery roller 41.
  • the protein raw material fiber 36 is overfed, and the protein raw material fiber 36 is provided between the delivery roller 41 and the take-up roller 42.
  • a relaxed state (not tensioned or tensioned) occurs.
  • a speed controller 46 is connected to the delivery roller 41 and the take-up roller 42.
  • the speed controller 46 is connected to a drive motor (not shown) provided on each of the delivery roller 41 and the winding roller 42.
  • the speed controller 46 is a computer including hardware such as a central processing unit (CPU), read only memory (ROM), and random access memory (RAM), and software such as a program stored in the ROM. .
  • the speed controller 46 regulates the delivery speed and / or the take-up speed as described above by controlling both the delivery roller 41 and the take-up roller 42, or any one of them.
  • the speed controller 46 constitutes speed adjusting means for adjusting at least one of the delivery speed of the delivery roller 41 and the take-up speed of the take-up roller 42.
  • the speed controller 46 may, for example, set the delivery speed and the delivery speed such that the delivery speed by the delivery roller 41 is an arbitrary ratio (relaxation factor) in the range of 1 to 3 times the take-up speed by the take-up roller 42. And / or take-up speed can be adjusted.
  • a high temperature heater 44 is attached in the high temperature heating furnace 43 of the high temperature heating and relaxing device 40.
  • a temperature controller 47 is connected to the high temperature heater 44.
  • the temperature controller 47 is a computer including hardware such as a CPU, a ROM, and a RAM, and software such as a program stored in the ROM.
  • the temperature controller 47 controls the temperature of the high temperature heating furnace 43 by controlling the high temperature heater 44.
  • Information on the temperature in the high temperature heating furnace 43 is input to the temperature controller 47 from a temperature sensor (not shown) provided in the high temperature heating furnace 43, and the temperature controller 47 controls the high temperature heater 44 based on the information. It is also good.
  • the temperature controller 47 constitutes temperature control means for controlling the heating temperature of the protein raw material fiber 36 in the high temperature heating furnace 43.
  • the temperature controller 47 controls the high temperature heater 44 such that the heating temperature in the high temperature heating furnace 43 is higher than the heating temperature in the drying furnace 17 of the drying device 4.
  • the temperature controller 47 can adjust the heating temperature of the protein raw material fiber 36 so as to be an arbitrary temperature in the range of 80 to 300 ° C., for example.
  • the speed controller 46 and the temperature controller 47 are separately shown, the present invention is not limited to such an aspect.
  • the speed controller 46 and the temperature controller 47 may be incorporated into an integrated controller, or a controller that controls the entire manufacturing apparatus 10 may be provided with functions equivalent to the speed controller 46 and the temperature controller 47. .
  • the high temperature heating and relaxing device 40 constitutes a heating means for heating the protein raw material fiber 36 and a relaxation and contraction means for relaxing and shrinking the protein raw material fiber 36 in the heated state.
  • the high-temperature heating and relaxing apparatus 40 is an apparatus that combines heating means and relaxation and contraction means.
  • the heating step and the relaxation and contraction step are simultaneously performed.
  • the high-temperature heating and relaxing apparatus 40 also serves as a means for adjusting the amount of relaxation of the protein raw material fiber 36.
  • this relaxation amount adjusting means is constituted by the above-mentioned speed adjusting means.
  • the high temperature thermal relaxation device 40 produces a protein fiber 50 having high tensile strength and high elongation.
  • the tensile strength and the elongation of the protein fiber can be optionally set by adjusting the draw ratio in the drawing means 26 of the drying device 4 and the contraction amount of the protein raw material fiber 36 in the high temperature heating relaxation device 40. It can be controlled.
  • a winder is provided on the downstream side of the high-temperature heating and relaxation device 40 in the traveling direction of the protein raw material fiber 36 and the protein fiber 50.
  • the protein fiber 50 is subjected to a shrink-proofing treatment in the high-temperature heat relaxation device 40 and then wound up by a winder to obtain the wound product 5.
  • the method of producing the protein fiber 50 using the production apparatus 10 will be described in more detail.
  • the protein raw material fiber 36 is spun, for example, by dry-wet spinning, using the above-described dope solution.
  • the protein raw material fiber 36 is drawn by the drawing means 26 (drawing process), and the protein raw material fiber 36 subjected to the drawing process is heated by the high-temperature heating relaxation device 40 (heating process). Relax and contract (relaxation step).
  • the drawing step includes a drawing ratio adjustment step of adjusting the drawing ratio of the protein raw material fiber 36. This process is performed by the first nip roller 13 and the second nip roller 14 described above and a controller that controls them.
  • the relaxation contraction step and the heating step are simultaneously performed in the high temperature heating and relaxing device 40.
  • the contraction amount adjustment step of adjusting the contraction amount of the protein raw material fiber 36 is performed .
  • the contraction amount adjustment step at least one of the relaxation amount of the protein raw material fiber 36 and the heating temperature of the protein raw material fiber 36 is adjusted.
  • the shrinkage adjustment process the protein raw fiber 36 is continuously delivered at a predetermined delivery speed, and the protein raw fiber 36 is continuously wound at a take-up speed slower than the delivery speed. Thereby, the protein raw fiber 36 is relaxed and contracted.
  • the contraction amount adjusting step the amount of relaxation of the protein raw material fiber 36 is adjusted by adjusting at least one of the delivery speed and the winding speed. Each of these steps is performed by the delivery roller 41, the winding roller 42, and the speed controller 46, and the high temperature heater 44 and the temperature controller 47 described above.
  • protein fibers for example, protein fibers containing spider silk fibroin
  • thermally shrunk protein fibers are improved in elongation according to the amount of shrinkage.
  • the mechanism of heat contraction of protein fiber may be considered as follows. First, it is due to the release of water in the fiber. In order to release the moisture, it is preferable that the heating temperature of the protein raw material fiber 36 be 80 ° C. or higher. Secondly, the internal stress generated by drawing or the like in the spinning process is caused by relaxation by heating. It is preferable that the heating temperature of the protein raw material fiber 36 be 180 ° C. or more in order to relieve the internal stress.
  • the heating temperature of the protein raw material fiber 36 in the heating step is preferably less than 180 ° C., and more preferably 80 ° C. or more and less than 180 ° C.
  • the speed of the delivery roller 41 and the winding roller 42 is kept unchanged, and the heating temperature is also kept constant.
  • protein fibers having constant strength and elongation can be stably produced.
  • the delivery roller 41 and the take-up roller 42 may be arbitrarily adjustable in speed, and / or the heating temperature may be optionally adjustable.
  • the heating step and the relaxing step may be performed separately if the protein raw material fiber 36 can be relaxed in a heated state. That is, the heating device may be a device separate and independent from the relaxation device. In that case, a relaxation device is provided downstream of the heating device (downstream in the traveling direction of the protein raw material fiber 36) so that the relaxation and contraction step is performed after the heating step.
  • the protein raw material fiber 36 in the heated state through the drawing process is relaxed and contracted to have high tensile strength and high elongation.
  • the protein fiber 50 can be easily manufactured.
  • the tensile strength and the elongation of the protein fiber 50 can be arbitrarily controlled.
  • the tensile strength and the elongation of the protein fiber 50 can be arbitrarily controlled.
  • the tensile strength and elongation of the protein fiber 50 can be arbitrarily controlled.
  • the protein material fiber 36 is relaxed and contracted by continuously feeding the protein material fiber 36 at a predetermined delivery speed and continuously winding it at a slower winding speed than the delivery speed. In addition, by adjusting at least one of the delivery speed and the take-up speed, the amount of relaxation of the protein raw material fiber 36 is controlled. By these steps, the productivity can be improved by the continuous production of the protein fiber 50.
  • the heating temperature of the protein raw material fiber 36 is less than 180 ° C., a decrease in tensile strength due to heating can be advantageously prevented.
  • the manufacturing process of the protein raw material fiber includes the drawing process
  • the heating and relaxing process is performed subsequently to the manufacturing process of the protein raw material fiber
  • the process from the dope solution to the production of the high tenacity fiber is performed in series. be able to.
  • the productivity of the high tenacity protein fiber 50 is improved.
  • the method for producing a protein fiber of the present disclosure described above is a processing method for processing a protein fiber produced through any known process, and includes a drawing process of drawing a protein fiber containing protein, and a protein obtained through the drawing process.
  • a method of processing a protein fiber comprising a heating step of heating the fiber, and a relaxation and contraction step which is performed simultaneously with or after the heating step and which relaxes and shrinks the protein fiber in a heated state by the heating step. You can think of it as
  • the amino acid sequence shown by SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes, further The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 11 is added to the N-terminus.
  • nucleic acid encoding PRT799 was synthesized.
  • the NdeI site at the 5 'end and the EcoRI site downstream of the stop codon were added to the nucleic acid.
  • the nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with NdeI and EcoRI, cut out, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector.
  • E. coli BLR (DE3) was transformed with the pET22b (+) expression vector containing a nucleic acid encoding PRT799.
  • the transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture broth was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that the OD 600 was 0.005.
  • the culture solution temperature was maintained 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 a production medium (Table 5 below) was added so that the OD 600 was 0.05.
  • the temperature of the culture solution was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the temperature of the culture solution was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of PRT799. Twenty hours after the addition of IPTG, the culture solution was centrifuged to recover the cells. SDS-PAGE was performed using cells prepared from the culture solution before IPTG addition and after IPTG addition, and the expression of PRT 799 was confirmed by the appearance of a band having a size corresponding to PRT 799 depending on IPTG addition.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the precipitate after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, 60 ° C. The solution was stirred for 30 minutes and dissolved. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Pure Chemical Industries, Ltd.). The white aggregated protein (PRT 799) obtained after dialysis was collected by centrifugation, the water was removed by a lyophilizer, and the lyophilized powder was collected.
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • Table 7 shows the test results of Examples 1 and 2 and Comparative Example 1 in which the stretching ratio was made the same, and the relaxation ratio and heating temperature were changed. The values of stress and elongation in Examples 1 and 2 are indicated by the ratio when Comparative Example 1 is 100%.
  • Table 8 shows the test results of Comparative Examples 1 to 3 which were conducted at the same temperature, without relaxation, but at different draw ratios. The values of stress and elongation in Comparative Examples 2 and 3 are shown as ratios when Comparative Example 1 is 100%.
  • Table 9 shows the test results of Examples 3 and 4 and Comparative Example 1 in which the draw ratio was changed, and the temperature was increased according to the contraction rate to cause relaxation.
  • the values of stress and elongation in Examples 3 and 4 are shown by the ratio when Comparative Example 1 is 100%.
  • protein fibers having high tensile strength and high elongation can be conveniently produced.

Abstract

L'invention concerne un procédé et un dispositif de production de fibres protéiques permettant de produire facilement des fibres protéiques présentant une grande résistance à la traction et un fort allongement. Le procédé de production de fibres protéiques comprend : une étape d'étirement au cours de laquelle des fibres en une matière première protéique (36) contenant une protéine sont étirées ; une étape de chauffage au cours de laquelle les fibres en une matière première protéique (36) qui ont subi l'étape d'étirement sont chauffées ; et une étape de détente et de contraction qui se déroule en même temps que l'étape de chauffage ou après cette dernière et au cours de laquelle les fibres en une matière première protéique (36) dans un état chauffé au cours de l'étape de chauffage sont détendues et amenées à se contracter.
PCT/JP2018/036532 2017-09-29 2018-09-28 Procédé et dispositif de production de fibres protéiques, procédé de traitement de fibres protéiques WO2019066053A1 (fr)

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WO2021060481A1 (fr) * 2019-09-27 2021-04-01 Ｓｐｉｂｅｒ株式会社 Procédé de fabrication de fibre protéique, procédé de fabrication de tissu de fibres protéiques et procédé anti-rétrécissement pour fibre protéique
CN113089153A (zh) * 2021-03-18 2021-07-09 清华大学 增强蛋白质纤维力学性能的方法及改性蛋白质纤维

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JP2009155774A (ja) * 2007-12-27 2009-07-16 Toray Ind Inc 繊維構造物およびその製造方法
JP2013506058A (ja) * 2009-09-28 2013-02-21 タフツ ユニバーシティー／トラスティーズ オブ タフツ カレッジ 延伸したシルクｅｇｅｌ繊維およびその製造方法
JP2014029054A (ja) * 2012-06-28 2014-02-13 Spiber Inc 原着タンパク質繊維及びその製造方法

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JPS4839371B1 (fr) * 1969-05-02 1973-11-24
JP2009155774A (ja) * 2007-12-27 2009-07-16 Toray Ind Inc 繊維構造物およびその製造方法
JP2013506058A (ja) * 2009-09-28 2013-02-21 タフツ ユニバーシティー／トラスティーズ オブ タフツ カレッジ 延伸したシルクｅｇｅｌ繊維およびその製造方法
JP2014029054A (ja) * 2012-06-28 2014-02-13 Spiber Inc 原着タンパク質繊維及びその製造方法

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021060481A1 (fr) * 2019-09-27 2021-04-01 Ｓｐｉｂｅｒ株式会社 Procédé de fabrication de fibre protéique, procédé de fabrication de tissu de fibres protéiques et procédé anti-rétrécissement pour fibre protéique
CN114521217A (zh) * 2019-09-27 2022-05-20 丝芭博株式会社 蛋白质纤维的制备方法、蛋白质纤维面料的制备方法以及蛋白质纤维的防缩加工方法
EP4036291A4 (fr) * 2019-09-27 2023-08-09 Spiber Inc. Procédé de fabrication de fibre protéique, procédé de fabrication de tissu de fibres protéiques et procédé anti-rétrécissement pour fibre protéique
CN113089153A (zh) * 2021-03-18 2021-07-09 清华大学 增强蛋白质纤维力学性能的方法及改性蛋白质纤维
CN113089153B (zh) * 2021-03-18 2022-05-24 清华大学 增强蛋白质纤维力学性能的方法及改性蛋白质纤维

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