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WO2018164190A1 - Synthetic fibroin fiber - Google Patents

Synthetic fibroin fiber Download PDF

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

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin

Definitions

  • the present invention relates to an artificial fibroin fiber.
  • Silk which is a kind of natural fibroin fiber, is known as a fiber that sufficiently satisfies the touch and has a high-quality feeling.
  • fibers used in clothing, bedding, etc. are required to have softness and heat retention. Therefore, silk used for clothing, bedding, and the like is subjected to, for example, shrink processing to increase bulkiness, thereby imparting flexibility and heat retention in some cases.
  • synthetic fibers such as polyester fibers, polyamide fibers, and acrylic fibers are generally used for clothing, bedding, etc., and these synthetic fibers have a shrinkage ratio of 40% or more when brought into contact with boiling water.
  • An object of the present invention is to provide an artificial fibroin fiber that has a sufficiently high shrinkage rate and can be manufactured safely.
  • the present invention relates to the following inventions, for example.
  • Shrinkage rate ⁇ 1 ⁇ (length of artificial fibroin fiber subjected to shrinking process including contact with water having less than boiling point / length of artificial fibroin fiber before performing the shrinking process) ⁇ ⁇ 100 (%)
  • the artificial fibroin fiber according to [1] wherein the modified fibroin is a modified spider silk fibroin.
  • an artificial fibroin fiber that has a sufficiently high shrinkage rate and can be manufactured safely.
  • the modified fibroin according to the present embodiment has a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing.
  • an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either one or both of the N-terminal side and the C-terminal side of the domain sequence.
  • the N-terminal sequence and the C-terminal sequence are not limited to these, but are typically regions having no amino acid motif repeat characteristic of fibroin and consisting of about 100 amino acids.
  • modified fibroin means an artificially produced fibroin (artificial fibroin).
  • the modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be a fibroin having the same amino acid sequence as that of naturally occurring fibroin.
  • Natural fibroin as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif.
  • a protein comprising a domain sequence to be processed.
  • the amino acid sequence of naturally-occurring fibroin may be used as it is, depending on the amino acid sequence of naturally-occurring fibroin.
  • the amino acid sequence may be modified (for example, the amino acid sequence may be modified by modifying the gene sequence of the cloned natural fibroin), and may be artificially designed and used independently of the natural fibroin. It may be synthesized (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).
  • domain sequence refers to a fibroin-specific crystal region (typically corresponding to the (A) n motif in the amino acid sequence) and an amorphous region (typically in the REP of the amino acid sequence).
  • (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
  • the number of amino acid residues of the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16 .
  • the ratio of the number of alanine residues to the total number of amino acid residues in the (A) n motif may be 40% or more, such as 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).
  • a plurality of (A) n motifs present in the domain sequence may be composed of at least seven alanine residues alone.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300, and may be an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • (A) n motif is preferably alanine residues is 83% or more to the total number of amino acid residues in the n motif, more preferably 86% or more, 90% or more Is more preferably 95% or more, particularly preferably 100% (meaning that it is composed of only alanine residues). It is preferable that at least seven of the (A) n motifs present in the domain sequence are composed of only alanine residues. Consists of only alanine residues means that (A) n motif is (A) n (A represents an alanine residue, n is an integer of 2 to 20, preferably 4 to 20, more preferably 4 to It represents an integer of 16).
  • the modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or a plurality of amino acid residues to the cloned gene sequence of naturally derived fibroin. It can be obtained by modifying the above. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
  • Naturally-derived fibroin is a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif.
  • Specific examples include fibroin produced by insects or spiders.
  • fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandaraina), Tengea (Antherea yamanai), ⁇ ⁇ (Antereaperyi), ⁇ ⁇ (Eriothyraminey) ), Silkworms (Samia cythia), chestnut worms (Caligula japonica), Chussa moth (Anthereaea mylitta), silkworms produced by silkworms such as Antheraea assamata, vespasam worms Examples include silk proteins.
  • fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).
  • Fibroin produced by spiders includes, for example, spiders belonging to the genus spider (Araneus spp.) Such as the spider spider, the spider spider, the red spider spider, and the bean spider, the genus spiders of the genus Araneus, the spider spider spider, the spider spider genus e Spiders, spiders such as spiders, spiders belonging to the genus Spider, spiders belonging to the genus Pronos, spiders belonging to the genus Trinofunda, such as Torinofundamas (genus Cyrtarachne) Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Spider (Ordgarius genus), such as the spiders, the spiders, and the spiders belonging to the genus Ordgarius Spiders belonging to the genus Argiope, such as the genus Argiope, spiders belonging to the genus Arachnura, such as the white-tailed spider, spiders belonging to the
  • Spiders belonging to the genus Azumigumi (Menosira), spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the common spider spider, the black spider spider, the genus Spider genus belonging to the genus Spider belonging to the genus (L) and the genus Spider belonging to the genus (L) Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops
  • Examples include spider silk protein.
  • the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.
  • fibroin produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin- 4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidolin 1 [derived from Nephila clavipes (GenBank accession number AAC4, amino acid sequence A04) U37520 (base sequence)), major sample spidroin 1 [La rodectus hesperus origin] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Nephila clavata origin] (GenBank accession number AAL32447, amino acid sequence 45, amino acid sequence) major amploidate
  • Naturally derived fibroin include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidin, sample, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION from sequences including INV as DIVISION among the sequence information registered in NCBI GenBank. It can be confirmed by extracting a character string of a specific product from the sequence, CDS, and a sequence in which the specific character string is described from SOURCE to TISSUE TYPE.
  • the modified fibroin according to the present embodiment may be a modified silk fibroin (a modified amino acid sequence of a silk protein produced by a silkworm), and a modified spider silk fibroin (an amino acid sequence of a spider silk protein produced by a spider). It may be modified).
  • the modified fibroin with a reduced content of glycine residues (modified fibroin according to the first embodiment), (A) the content of the n motif is reduced.
  • the modified fibroin according to the first embodiment has an amino acid sequence in which the content of a glycine residue is reduced as compared with a naturally occurring fibroin. It can be said that the modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with naturally occurring fibroin.
  • the modified fibroin according to the first embodiment has at least a domain sequence selected from GGX and GPGXX in REP (where X represents an amino acid residue other than glycine) as compared to naturally occurring fibroin.
  • One motif sequence may have an amino acid sequence corresponding to the substitution of one glycine residue in at least one or a plurality of the motif sequences with another amino acid residue.
  • the ratio of the motif sequence in which the above-described glycine residue is substituted with another amino acid residue may be 10% or more with respect to the entire motif sequence.
  • the modified fibroin according to the first embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and (A) n motif located on the most C-terminal side from the domain sequence.
  • Z is the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the domain sequence to the C-terminal of the domain sequence.
  • z / w is It may have an amino acid sequence that is 30% or more, 40% or more, 50% or more, or 50.9% or more.
  • the modified fibroin according to the first embodiment is obtained by substituting one glycine residue of the GGX motif with another amino acid residue. It is preferable that the content ratio of the amino acid sequence consisting of XGX is increased.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, and preferably 10% or less. More preferably, it is still more preferably 6% or less, still more preferably 4% or less, and particularly preferably 2% or less.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.
  • an amino acid sequence consisting of XGX is extracted from all REPs included in the sequence excluding the sequence from the domain motif (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • z / w (%) can be calculated by dividing z by w.
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, and further preferably 58.7% or more. 70% or more is even more preferable, and 80% or more is even more preferable. Although there is no restriction
  • the modified fibroin according to the first embodiment for example, from the cloned gene sequence of naturally-occurring fibroin, at least a part of the base sequence encoding a glycine residue is substituted to encode another amino acid residue. It can be obtained by modifying. At this time, one glycine residue in GGX motif and GPGXX motif may be selected as a glycine residue to be modified, or substitution may be performed so that z / w is 50.9% or more.
  • an amino acid sequence satisfying the above-described aspect can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence corresponding to the insertion and / or addition may be modified.
  • the other amino acid residue is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( M) hydrophobic amino acid residues such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and glutamic acid (E) residues are preferred, and valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residue is more preferable, and glutamine (Q) residue is more preferable.
  • modified fibroin As more specific examples of the modified fibroin according to the first embodiment, (1-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (1-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more of sequence identity with the amino acid sequence shown in No. 3, SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12.
  • the modified fibroin (1-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin.
  • the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence represented by SEQ ID NO: 3, in which every two (A) n motifs are deleted from the N-terminal side to the C-terminal side, and further before the C-terminal sequence.
  • One [(A) n motif-REP] is inserted into the.
  • the amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4.
  • the amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
  • the value of z / w in the amino acid sequence represented by SEQ ID NO: 1 is 46.8%.
  • the z / w values in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 12 are 58.7%, 70.1%, 66.1% and 70.0%.
  • the value of x / y at the ratio of the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 (described later) 1: 1.8 to 11.3 is: 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.
  • the modified fibroin (1-i) may consist of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin (1-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin of (1-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (1-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules.
  • affinity tag include a histidine tag (His tag).
  • His tag is a short peptide with about 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography) Can be used.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 5 (amino acid sequence containing a His tag).
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • an “epitope tag” using an antigen-antibody reaction can also be used.
  • a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound.
  • HA peptide sequence of hemagglutinin of influenza virus
  • myc tag peptide sequence of hemagglutinin of influenza virus
  • FLAG tag peptide sequence of hemagglutinin of influenza virus
  • a tag sequence that can be separated with a specific protease can also be used.
  • the modified fibroin from which the tag sequence has been separated can also be recovered.
  • modified fibroin containing the tag sequence examples include (1-iii) the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (1-iv) SEQ ID NO: 8 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively.
  • amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
  • the modified fibroin (1-iii) may be composed of the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin (1-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin of (1-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (1-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the modified fibroin according to the second embodiment has an amino acid sequence in which the domain sequence has a reduced content of (A) n motif compared to naturally occurring fibroin. It can be said that the domain sequence of the modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) n motifs as compared to naturally occurring fibroin.
  • the modified fibroin according to the second embodiment may have an amino acid sequence corresponding to 10% to 40% deletion of the (A) n motif from naturally occurring fibroin.
  • the modified fibroin according to the second embodiment has a domain sequence of 1 to 3 (A) n motifs at least from the N-terminal side to the C-terminal side as compared with naturally-occurring fibroin ( A) It may have an amino acid sequence corresponding to deletion of the n motif.
  • the modified fibroin according to the second embodiment has a domain sequence that is at least two consecutive from the N-terminal side to the C-terminal side compared to naturally-occurring fibroin (A) deletion of the n motif, and It may have an amino acid sequence corresponding to the deletion of one (A) n motif repeated in this order.
  • the modified fibroin according to the second embodiment has an amino acid sequence corresponding to the deletion of every two (A) n motifs at least every two domain sequences from the N-terminal side to the C-terminal side. There may be.
  • the modified fibroin according to the second embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [[( A) The number of amino acid residues in the REP of the n motif-REP] unit is sequentially compared, and when the number of amino acid residues in the REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is The maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units that are 1.8 to 11.3 is x, and the total number of amino acid residues in the domain sequence is When y, x / y may have an amino acid sequence of 20% or more, 30% or more, 40% or more, or 50% or more.
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin.
  • the domain sequence is from the N-terminal side (left side): (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called n motif.
  • FIG. 1 includes pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison), pattern 4 (first REP and Comparison of the second REP).
  • pattern 1 compare between the first REP and the second REP, and comparison between the third REP and the fourth REP
  • pattern 2 comparison between the first REP and the second REP, and 4th REP and 5th REP
  • pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison
  • pattern 4 first REP and Comparison of the second REP
  • the number of amino acid residues of each REP in the two adjacent [(A) n motif-REP] units selected is compared.
  • each pattern the number of all amino acid residues of two adjacent [(A) n motif-REP] units indicated by solid lines is added (not only REP but also (A) the number of amino acid residues of the n motif. is there.). Then, the total value added is compared, and the total value (maximum value of the total value) of the pattern having the maximum total value is set as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
  • x / y (%) can be calculated by dividing x by the total number of amino acid residues y of the domain sequence.
  • x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, and 70% or more. Is more preferably 75% or more, still more preferably 80% or more.
  • x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, and 70% or more. Is more preferably 75% or more, still more preferably 80% or more.
  • limiting in particular in the upper limit of x / y For example, you may be 100% or less.
  • x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x / y / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.
  • x / y is 46.4% Preferably, it is 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, It is especially preferable that it is 80% or more.
  • x / y is 46.4%
  • it is 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, It is especially preferable that it is 80% or more.
  • x / y in naturally derived fibroin will be described.
  • 663 types of fibroin (of which 415 types were derived from spiders) were extracted.
  • x / y is calculated from the amino acid sequence of naturally derived fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above-described calculation method.
  • FIG. 3 shows the results when the jagged ratio is 1: 1.9 to 4.1.
  • the horizontal axis indicates x / y (%), and the vertical axis indicates frequency.
  • x / y in naturally derived fibroin is less than 64.2% (the highest, 64.14%).
  • the modified fibroin according to the second embodiment includes, for example, one or a plurality of sequences encoding the n motif so that x / y is 64.2% or more from the cloned gene sequence of natural fibroin (A) Can be obtained by deleting.
  • an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to this may be modified.
  • modified fibroin As a more specific example of the modified fibroin according to the second embodiment, (2-i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in No. 2, SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12.
  • the modified fibroin (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 2 has the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin deleted from the N-terminal side to the C-terminal side every two (A) n motifs Furthermore, one [(A) n motif-REP] is inserted in front of the C-terminal sequence.
  • the amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting all GGX in REP of the amino acid sequence shown in SEQ ID NO: 2 with GQX.
  • the amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4.
  • the amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
  • the value of x / y of the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally-occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 4 is 93.4%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 12 is 89.8%.
  • the z / w values in the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 are 46.8%, 56.2%, 70.1% and 66. respectively. 1% and 80.4%.
  • the modified fibroin (2-i) may be composed of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and from the N-terminal side to the C-terminal side
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other
  • x / y is 64.2% or more, where x is the maximum total value of the total number of bases and y is the total number of amino acid residues in the domain sequence.
  • the above-described modified fibroin may contain the above-described tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin containing the tag sequence examples include (2-iii) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 7 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively.
  • amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
  • the modified fibroin (2-iii) may be composed of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (2-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and from the N-terminal side to the C-terminal side.
  • the other X is the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3.
  • x / y is preferably 64.2% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the domain sequence has a reduced content of glycine residues in addition to the reduced content of the (A) n motif compared to the naturally derived fibroin.
  • the domain sequence of the modified fibroin is different from that of naturally occurring fibroin in addition to at least one or more glycine residues in REP. It can be said to have an amino acid sequence corresponding to substitution with an amino acid residue. That is, the modified fibroin having the characteristics of the modified fibroin according to the first embodiment described above and the modified fibroin according to the second embodiment. Specific aspects and the like are as described in the modified fibroin according to the first and second embodiments.
  • modified fibroin More specific examples of the modified fibroin according to the third embodiment include (3-i) an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, (3-ii) SEQ ID NO: 4, SEQ ID NO: Examples thereof include modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 10 or SEQ ID NO: 12. Specific embodiments of the modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.
  • the modified fibroin according to the fourth embodiment has a domain sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues having a large hydrophobicity index as compared with naturally occurring fibroin, And / or having an amino acid sequence that locally includes a region having a large hydrophobicity index corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index in REP.
  • the region where the hydrophobic index is locally large is preferably composed of 2 to 4 amino acid residues.
  • the amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). More preferably, it is a residue.
  • the modified fibroin according to the fourth embodiment has one or more amino acid residues in REP substituted with amino acid residues having a large hydrophobicity index and / or in REP compared to naturally occurring fibroin.
  • substitution, deletion or insertion of one or more amino acid residues as compared with naturally occurring fibroin And / or there may be amino acid sequence modifications corresponding to the addition.
  • the modified fibroin according to the fourth embodiment includes, for example, one or more hydrophilic amino acid residues (for example, amino acid residues whose hydrophobicity index is negative) in REP from the cloned gene sequence of naturally-occurring fibroin. It can be obtained by substituting with a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP.
  • hydrophilic amino acid residues for example, amino acid residues whose hydrophobicity index is negative
  • one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to insertion of, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin and / or one or more hydrophobic amino acids in REP
  • the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.
  • the modified fibroin according to the fourth embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side (A) from the n motif of the domain sequence.
  • the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence obtained by removing the sequence up to the C-terminal from the domain sequence.
  • P, and (A) where the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is q / Q may have an amino acid sequence of 6.2% or more.
  • hydrophobicity index of amino acid residues As for the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of bio.p. 7”. 105-132). Specifically, the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
  • a sequence obtained by removing the sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m to the most C-terminal side from the domain (A) n motif to the C terminus of the domain sequence. (Hereinafter referred to as “array A”).
  • array A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated.
  • the average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in the four consecutive amino acid residues by 4 (number of amino acid residues).
  • the average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of hydrophobicity index is 2.6 or more”, it should be included as one amino acid residue in the region. become.
  • the total number of amino acid residues contained in the region is p.
  • the total number of amino acid residues contained in sequence A is q.
  • the average value of the hydrophobicity index of four consecutive amino acid residues is 2
  • p / q is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, and 20% or more. It is still more preferable that it is 30% or more.
  • the upper limit of p / q is not particularly limited, but may be 45% or less, for example.
  • the modified fibroin according to the fourth embodiment includes, for example, one or a plurality of hydrophilic amino acid residues in REP (for example, an amino acid sequence of a cloned naturally-derived fibroin so as to satisfy the above p / q condition) Substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or one or more hydrophobic amino acids during REP By inserting a residue, it can be obtained by locally modifying the amino acid sequence including a region having a large hydrophobicity index.
  • an amino acid sequence satisfying the above p / q conditions can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index and / or one or more amino acid residues in REP.
  • modifications corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. .
  • the amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
  • modified fibroin As another specific example of the modified fibroin according to the fourth embodiment, (4-i) the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, or (4-ii) SEQ ID NO: 15, Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.
  • the modified fibroin (4-i) will be described.
  • the amino acid sequence shown in SEQ ID NO: 14 is an amino acid sequence in which alanine residues in the (A) n motif of (A) naturally derived fibroin are deleted so that the number of consecutive alanine residues is five.
  • the amino acid sequence shown in SEQ ID NO: 15 is inserted into the amino acid sequence shown in SEQ ID NO: 14 by two amino acid sequences (VLI) each consisting of 3 amino acid residues for every other REP, and shown in SEQ ID NO: 14. A part of amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence is almost the same.
  • the amino acid sequence shown in SEQ ID NO: 16 is obtained by inserting two alanine residues on the C-terminal side of each (A) n motif with respect to the amino acid sequence shown in SEQ ID NO: 14, and further, some glutamine (Q) residues. A group is substituted with a serine (S) residue, and a part of amino acids on the C-terminal side is deleted so as to be approximately the same as the molecular weight of the amino acid sequence represented by SEQ ID NO: 14.
  • the amino acid sequence represented by SEQ ID NO: 17 is obtained by inserting one amino acid sequence (VLI) consisting of 3 amino acid residues every other REP to the amino acid sequence represented by SEQ ID NO: 16.
  • the amino acid sequence shown in SEQ ID NO: 18 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues into the amino acid sequence shown in SEQ ID NO: 16 every other REP.
  • the modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18.
  • the modified fibroin (4-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18.
  • the modified fibroin of (4-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (4-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, and is located at the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the above-mentioned modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin containing a tag sequence (4-iii) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (4-iv) SEQ ID NO: 19, SEQ ID NO: 20 or A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21 can be mentioned.
  • amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 are the amino acid sequences represented by SEQ ID NO: 5 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18, respectively (His tag). Including a sequence and a hinge sequence).
  • the modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin (4-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (4-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (4-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the modified fibroin according to the present embodiment includes, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing the nucleic acid.
  • the method for producing the nucleic acid encoding the modified fibroin is not particularly limited.
  • the nucleic acid is produced by a method such as amplification by polymerase chain reaction (PCR), cloning, modification by genetic engineering techniques, or chemical synthesis. can do.
  • the method for chemically synthesizing nucleic acids is not particularly limited. For example, based on the amino acid sequence information of fibroin obtained from the NCBI web database, etc., AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) A gene can be chemically synthesized by a method of linking oligonucleotides that are synthesized automatically by PCR or the like.
  • a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N terminus of the above amino acid sequence is synthesized. May be.
  • Regulatory sequences are sequences that control the expression of modified fibroin in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected depending on the type of host.
  • an inducible promoter that functions in the host cell and can induce expression of the modified fibroin may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value.
  • the type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like.
  • a vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like.
  • microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • a prokaryotic host is used as a vector for introducing a nucleic acid encoding a modified fibroin
  • a prokaryotic host for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like can be mentioned.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like.
  • Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma and the like.
  • examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC 37115) and YEp24 (ATCC 37051).
  • a method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • electroporation method electroporation method
  • spheroplast method protoplast method
  • lithium acetate method competent method, and the like.
  • a method for expressing a nucleic acid by a host transformed with an expression vector in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd edition, etc. .
  • the modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting from the culture medium.
  • the method for culturing a host in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.
  • Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism.
  • Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc.
  • Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source examples include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • Isolation and purification of the expressed modified fibroin can be performed by a commonly used method.
  • the host cells are recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a manton. A host cell is crushed with a Gaurin homogenizer, dynomill, etc., and a cell-free extract is obtained.
  • a method usually used for isolation and purification of modified fibroin that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method by DE, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), or a resin such as S-Sepharose FF (manufactured by Pharmacia) Electrophoresis such as cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Use methods such as law alone or in combination It is possible to obtain a purified product.
  • a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA
  • the modified fibroin when expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to collect the modified fibroin insoluble substance as a precipitate fraction.
  • the recovered insoluble form of modified fibroin can be solubilized with a protein denaturant.
  • a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.
  • the modified fibroin when secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture with a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the artificial fibroin fiber according to the present embodiment can be produced by a known spinning method. That is, for example, when producing an artificial fibroin fiber containing a modified fibroin as a main component, first, the modified fibroin produced according to the above-described method is converted into dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), Alternatively, it is added to a solvent such as hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator and dissolved to prepare a dope solution. Next, the target artificial fibroin fiber can be obtained by spinning using this dope solution by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning. Preferred spinning methods include wet spinning or dry wet spinning.
  • FIG. 5 is an explanatory view schematically showing an example of a spinning device for producing artificial fibroin fibers.
  • a spinning device 10 shown in FIG. 5 is an example of a spinning device for dry and wet spinning, and includes an extrusion device 1, an undrawn yarn production device 2, a wet heat drawing device 3, and a drying device 4.
  • the dope solution 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8.
  • the dope solution may be filled into a cylinder and extruded from a nozzle using a syringe pump.
  • the extruded dope liquid 6 is supplied into the coagulating liquid 11 in the coagulating liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulated body is formed.
  • the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn.
  • the draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14.
  • the stretched fibrous solidified body is supplied to the drying device 4 and dried in the yarn path 22, and an artificial fibroin fiber is obtained as the wound thread body 5.
  • Reference numerals 18a to 18g denote thread guides.
  • the coagulation liquid 11 may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone.
  • the coagulation liquid 11 may appropriately contain water.
  • the temperature of the coagulation liquid 11 is preferably 0 to 30 ° C.
  • the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and 1.4 to 4.0 ml / hour More preferably it is time.
  • the distance through which the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) has only to be a length that allows efficient desolvation, for example, 200 to 500 mm. It is.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min.
  • the residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes.
  • stretching pre-stretching
  • the coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as necessary.
  • the stretching performed when obtaining the artificial fibroin fiber for example, dry stretching is also adopted in addition to the pre-stretching performed in the coagulating liquid tank 20 and the wet heat stretching performed in the stretching bath 21.
  • Wet and hot stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, and in steam heating.
  • the temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C.
  • undrawn yarn or predrawn yarn
  • Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C.
  • an undrawn yarn or predrawn yarn
  • an undrawn yarn can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching.
  • wet heat stretching and dry heat stretching can be appropriately combined.
  • the final draw ratio of the lower limit of the undrawn yarn (or predrawn yarn) is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times. Above, 7 times or more, 8 times or more, 9 times or more, and upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less 12 times or less, 11 times or less, and 10 times or less.
  • the artificial fibroin fiber according to this embodiment is obtained by spinning the above-described modified fibroin and contains the above-described modified fibroin as a main component.
  • the artificial fibroin fiber according to this embodiment preferably has a shrinkage rate defined by the following formula exceeding 7%. Thereby, it can shrink
  • Shrinkage rate ⁇ 1 ⁇ (length of artificial fibroin fiber subjected to shrinkage processing including contact with water having less than boiling point / length of artificial fibroin fiber before being subjected to shrinkage processing) ⁇ ⁇ 100 (%)
  • the shrinkage rate of the artificial fibroin fiber according to this embodiment is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, and further preferably 40% or more. More preferably, it is 48% or more, still more preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more.
  • the upper limit of shrinkage is usually 80% or less.
  • Shrinkage processing includes contacting artificial fibroin fiber with water having a boiling point (hereinafter also referred to as “contacting step”).
  • the shrinking process may further include drying the artificial fibroin fiber after contact with water (after the contact step) (hereinafter also referred to as “drying step”).
  • the temperature of the water to be brought into contact with the artificial fibroin fiber in the contact step may be less than the boiling point. Thereby, the handleability, the workability of shrinkage processing, and the like are improved. Further, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit value of the water temperature is preferably 10 ° C or higher, more preferably 40 ° C or higher, and further preferably 70 ° C or higher. . The upper limit of the water temperature is preferably 90 ° C. or lower.
  • the method for bringing water into contact with the artificial fibroin fiber is not particularly limited.
  • a method of immersing the artificial fibroin fiber in water a method of spraying water on the artificial fibroin fiber at room temperature or in a heated steam state, and the high humidity where the artificial fibroin fiber is filled with water vapor Examples include exposure to the environment.
  • the method of immersing the artificial fibroin fiber in water is preferable in the contact step because the shrinkage time can be effectively shortened and the processing equipment can be simplified.
  • the contact step when the artificial fibroin fiber is brought into contact with water in a relaxed state, the artificial fibroin fiber may not only contract but also shrink like a wave.
  • the contact step is performed in a state where the artificial fibroin fiber is not relaxed, for example, the artificial fibroin fiber is brought into contact with water below the boiling point while being tensioned (pulled) in the fiber axis direction. May be.
  • the drying step is a step of drying the artificial fibroin fiber that has undergone the contact step. Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known drying equipment of contact type or non-contact type can be used.
  • the drying temperature is not limited as long as it is lower than the temperature at which, for example, the protein contained in the artificial fibroin fiber is decomposed or the artificial fibroin fiber is thermally damaged.
  • the temperature is in the range of 20 to 150 ° C., and the temperature is preferably in the range of 50 to 100 ° C. When the temperature is in this range, the artificial fibroin fiber is dried more quickly and efficiently without thermal damage of the artificial fibroin fiber or degradation of the protein contained in the artificial fibroin fiber.
  • the drying time is appropriately set according to the drying temperature or the like. For example, a time during which the influence on the quality and physical properties of the artificial fibroin fiber due to overdrying can be eliminated as much as possible is adopted.
  • the artificial fibroin fiber according to the present invention can be highly contracted, for example, the highly contracted artificial fibroin fiber can be obtained through the above-described shrinking process (contacting step and drying step as necessary).
  • the high-shrinkage artificial fibroin fiber according to this embodiment has a shrinkage rate defined as described above exceeding 7%.
  • the shrinkage ratio of the high shrinkage artificial fibroin fiber is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, and still more preferably 40% or more, More preferably, it is 48% or more, particularly preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more.
  • the upper limit of shrinkage is usually 80% or less.
  • FIG. 6 is an explanatory view schematically showing an example of an apparatus for shrinking an artificial fibroin fiber.
  • the apparatus 40 shown in FIG. 6 includes a feed roller 42 that feeds out artificial fibroin fibers, a winder 44 that winds up the highly shrinkable artificial fibroin fibers 38, a water bath 46 that performs a contact step, and a dryer 48 that performs a drying step. , And is configured.
  • the feed roller 42 can be attached with a wound product of the artificial fibroin fiber 36, and the artificial fibroin fiber 36 is continuously formed from the wound product of the artificial fibroin fiber 36 by rotation of an electric motor (not shown). Can be delivered automatically and automatically.
  • the winder 44 can continuously and automatically wind up the high shrinkage artificial fibroin fiber 38 produced through the contact step and the drying step by the rotation of an electric motor (not shown). ing.
  • the feeding speed of the artificial fibroin fiber 36 by the feed roller 42 and the winding speed of the highly shrinkable artificial fibroin fiber 38 by the winder 44 can be controlled independently of each other.
  • the water bath 46 and the dryer 48 are arranged side by side between the feed roller 42 and the winder 44 on the upstream side and the downstream side in the feeding direction of the artificial fibroin fiber 36.
  • the apparatus 40 shown in FIG. 6 has relay rollers 50 and 52 that relay the artificial fibroin fiber 36 that travels from the feed roller 42 toward the winder 44.
  • the water bath 46 has a heater 54, and hot water 47 heated by the heater 54 is accommodated in the water bath 46.
  • a tension roller 56 is installed in the water bath 46 so as to be immersed in the hot water 47.
  • the artificial fibroin fiber 36 fed out from the feed roller 42 travels toward the winder 44 while being immersed in the hot water 47 while being wound around the tension roller 56 in the water bath 46. It has become.
  • the immersion time of the artificial fibroin fiber 36 in the hot water 47 is appropriately controlled according to the traveling speed of the artificial fibroin fiber 36.
  • the dryer 48 has a pair of hot rollers 58.
  • the pair of hot rollers 58 can be wound around the artificial fibroin fiber 36 that moves away from the water bath 46 and travels toward the winder 44 side.
  • the artificial fibroin fiber 36 immersed in the hot water 47 in the water bath 46 is heated by the pair of hot rollers 58 in the dryer 48 and dried, and then sent out further toward the winder 44. It has become.
  • the apparatus 40 When manufacturing the high-shrinkage artificial fibroin fiber 38 using the apparatus 40 having such a structure, first, for example, winding of the artificial fibroin fiber 36 spun using the spinning apparatus 10 shown in FIG. The circulation is mounted on the feed roller 42. Next, the artificial fibroin fiber 36 is continuously fed from the feed roller 42 and immersed in hot water 47 in the water bath 46. At this time, for example, the winding speed of the winder 44 is set slower than the feeding speed of the feed roller 42. Thereby, since the artificial fibroin fiber 36 is contracted by contact with the hot water 47 in a state where the artificial fibroin fiber 36 is tensioned so as not to relax between the feed roller 42 and the winder 44, occurrence of crimping can be prevented.
  • the artificial fibroin fiber 36 contracted in the hot water 47 in the water bath 46 is heated by a pair of hot rollers 58 of the dryer 48.
  • the contracted artificial fibroin fiber 36 is dried to obtain a highly contracted artificial fibroin fiber 38.
  • the artificial fibroin fiber 36 can be further contracted, and the length can be kept unchanged.
  • the obtained high-shrinkage artificial fibroin fiber 38 is wound up by a winder 44 to obtain a wound product of the high-shrinkage artificial fibroin fiber 38.
  • the drying means is constituted by the dry heat plate 64.
  • the intended high-shrinkage artificial fibroin fiber 38 can be manufactured automatically and continuously and extremely easily.
  • FIG. 7 is an explanatory view schematically showing another example of an apparatus for shrinking artificial fibroin fibers.
  • FIG. 7A shows a processing apparatus that performs the contact step provided in the apparatus
  • FIG. 7B shows a drying apparatus that performs the drying step provided in the apparatus.
  • the apparatus shown in FIG. 7 includes a processing device 60 that performs a contact step with respect to the artificial fibroin fiber 36, and a drying device 62 that dries the artificial fibroin fiber 36 subjected to the contact step with the processing device 60.
  • a processing device 60 that performs a contact step with respect to the artificial fibroin fiber 36
  • a drying device 62 that dries the artificial fibroin fiber 36 subjected to the contact step with the processing device 60.
  • the processing device 60 shown in FIG. 7A omits the dryer 48 from the device 40 shown in FIG. 6, and replaces the feed roller 42, the water bath 46, and the winder 44 with the artificial fibroin.
  • the fiber 36 has a structure in which the fibers 36 are arranged in order from the upstream side to the downstream side in the traveling direction.
  • the artificial fibroin fiber 36 fed from the feed roller 42 is immersed in hot water 47 in the water bath 46 and contracted before being wound up by the winder 44. .
  • the artificial fibroin fiber 36 contracted in the hot water 47 is configured to be wound up by the winder 44.
  • the 7B includes a feed roller 42 and a winder 44, and a dry heat plate 64.
  • the dry heat plate 64 is disposed between the feed roller 42 and the winder 44 so that the dry heat surface 66 contacts the artificial fibroin fiber 36 and extends along the traveling direction.
  • the artificial fibroin fiber 36 can be further shrunk by controlling the ratio of the feed speed of the feed roller 42 and the winding speed of the winder 44, for example. It is also possible not to change the length.
  • the artificial fibroin fiber 36 is shrunk by the processing device 60, and then the artificial fibroin fiber 36 is dried by the drying device 62.
  • High shrinkage man-made fibroin fiber 38 can be produced.
  • the processing apparatus 60 may be configured with only the water bath 46 by omitting the feed roller 42 and the winder 44 from the processing apparatus 60 shown in FIG.
  • high-shrinkage artificial fibroin fibers are manufactured in a so-called batch system.
  • Modified fibroin having the amino acid sequence represented by SEQ ID NO: 7 (PRT399), modified fibroin having the amino acid sequence represented by SEQ ID NO: 8 (PRT380), modified fibroin having the amino acid sequence represented by SEQ ID NO: 9 (PRT410), SEQ ID NO: A modified fibroin (PRT799) having the amino acid sequence shown by 13 was designed.
  • the amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin, wherein (A) n motif is deleted every two from the N-terminal side to the C-terminal side. Further, an amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminal to the amino acid sequence in which one [(A) n motif-REP] is inserted before the C-terminal sequence. .
  • amino acid sequence represented by SEQ ID NO: 8 is represented by SEQ ID NO: 5 at the N-terminal with respect to the amino acid sequence in which all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin are replaced with GQX. Added amino acid sequence (including His tag).
  • the amino acid sequence represented by SEQ ID NO: 9 is the amino acid sequence represented by SEQ ID NO: 5 at the N-terminus (with His tag) compared to the amino acid sequence in which all GGXs in REP of the amino acid sequence represented by SEQ ID NO: 2 are replaced with GQX Included).
  • the amino acid sequence shown in SEQ ID NO: 13 is a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted).
  • the amino acid sequence represented by SEQ ID NO: 5 (including the His tag) is added to the N-terminus of the amino acid sequence in which the His tag is added to the C-terminus of the sequence obtained by repeating 4 times.
  • the nucleic acids encoding the four types of designed modified fibroin were synthesized respectively.
  • the nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon.
  • These four types of nucleic acids were cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with the obtained pET-22b (+) expression vector.
  • the transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium (Table 2) containing ampicillin so that the OD 600 was 0.005.
  • the culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
  • the seed culture solution was added to a jar fermenter to which 500 ml of production medium (Table 3 below) was added so that the OD 600 was 0.05.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration.
  • 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target modified fibroin.
  • the culture solution was centrifuged, and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of a band of a size corresponding to the target modified fibroin depending on the addition of IPTG Expression was confirmed.
  • the washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 60 ° C. And stirred for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). White aggregated protein obtained after dialysis was collected by centrifugation. Water was removed from the recovered aggregated protein with a freeze dryer to obtain the desired modified fibroin freeze-dried powder.
  • 8M guanidine buffer 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • spun and drawn artificial fibroin fibers were produced by dry and wet spinning using a spinning device similar to the spinning device 10 shown in FIG.
  • the spinning device used is a spinning device 10 shown in FIG. 5, and a second undrawn yarn producing device (a first bath) and a wet heat drawing device 3 (third bath) between the undrawn yarn producing device 2 (first bath) and the second undrawn yarn producing device (3rd bath) A second bath).
  • the dry and wet spinning conditions are as follows. Extrusion nozzle diameter: 0.2 mm Liquid and temperature in the first to third baths: see Table 4 Total draw ratio: see Table 4 Drying temperature: 60 ° C.
  • a plurality of artificial fibroin fibers for testing each having a length of 30 cm were cut out from the wound product of artificial fibroin fibers obtained in Production Examples 1 to 19.
  • the plurality of artificial fibroin fibers were bundled to obtain an artificial fibroin fiber bundle having a fineness of 150 denier.
  • 0.8 g of lead weight was attached to each artificial fibroin fiber bundle, and in this state, each artificial fibroin fiber bundle was immersed in water having a temperature shown in Tables 5 to 8 for 10 minutes (shrinkage processing 1). Thereafter, the length of each artificial fibroin fiber bundle was measured in water.
  • the artificial fibroin fiber according to the present invention has a sufficiently high shrinkage rate.

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Abstract

The present invention pertains to a synthetic fibroin fiber which contains a modified fibroin and for which a shrinkage rate, defined by the formula below, exceeds 7%. Shrinkage rate = {1 − the length of a synthetic fibroin fiber which has undergone shrinkage processing including bringing the fiber into contact with water below the boiling point/the length of the synthetic fibroin fiber prior to undergoing the shrinkage processing} × 100(%)

Description

人造フィブロイン繊維Artificial fibroin fiber
　本発明は、人造フィブロイン繊維に関する。 The present invention relates to an artificial fibroin fiber.
　一般に、衣料、寝具等に使用される繊維には、高い肌触り性が求められる。肌触り性を充分に満足し、且つ高級感のある繊維として、天然のフィブロイン繊維の１種であるシルク等が知られている。 In general, fibers used for clothing, bedding, etc. are required to have a high touch. Silk, which is a kind of natural fibroin fiber, is known as a fiber that sufficiently satisfies the touch and has a high-quality feeling.
　また、衣料、寝具等に使用される繊維には、柔らかさ及び保温性等も同時に求められる。そのため、衣料、寝具等に用いられるシルクは、例えば、収縮加工が施されて嵩高性が高められ、それによって柔軟性及び保温性が付与される場合がある。 Also, fibers used in clothing, bedding, etc. are required to have softness and heat retention. Therefore, silk used for clothing, bedding, and the like is subjected to, for example, shrink processing to increase bulkiness, thereby imparting flexibility and heat retention in some cases.
　一方、例えば、衣料、寝具等には、ポリエステル繊維、ポリアミド繊維及びアクリル繊維等の合成繊維が汎用的に使用されおり、これらの合成繊維は、沸騰水に接触させることで４０％以上の収縮率を実現している（特許文献１）。 On the other hand, for example, synthetic fibers such as polyester fibers, polyamide fibers, and acrylic fibers are generally used for clothing, bedding, etc., and these synthetic fibers have a shrinkage ratio of 40% or more when brought into contact with boiling water. (Patent Document 1).
特開２００９－１２１００３号公報JP 2009-112003 A
　しかしながら、シルクは、水だけとの接触では収縮率が数％程度でしかなかった。 However, silk contracted only a few percent when contacted with water alone.
　また、特許文献１に記載された収縮方法は、高温の沸騰水を取り扱うために大きな危険が伴うものであった。 Further, the shrinkage method described in Patent Document 1 is accompanied by a great danger in handling hot boiling water.
　本発明は、充分に高い収縮率を有し、かつ安全に製造が可能な人造フィブロイン繊維を提供することを目的とする。 An object of the present invention is to provide an artificial fibroin fiber that has a sufficiently high shrinkage rate and can be manufactured safely.
　本発明は、例えば、以下の各発明に関する。
［１］
　改変フィブロインを含み、かつ下記式で定義される収縮率が７％を超える、人造フィブロイン繊維。
　収縮率＝｛１－（沸点未満の水に接触させることを含む収縮加工を施した人造フィブロイン繊維の長さ／上記収縮加工を施す前の人造フィブロイン繊維の長さ）｝×１００（％）
［２］
　上記改変フィブロインが、改変クモ糸フィブロインである、［１］に記載の人造フィブロイン繊維。
［３］
　上記水の温度が、１０～９０℃である、［１］又は［２］に記載の人造フィブロイン繊維。
［４］
　上記収縮加工が、上記水との接触後に乾燥させることを含む、［１］～［３］のいずれかに記載の人造フィブロイン繊維。 The present invention relates to the following inventions, for example.
[1]
An artificial fibroin fiber containing a modified fibroin and having a shrinkage rate defined by the following formula exceeding 7%.
Shrinkage rate = {1− (length of artificial fibroin fiber subjected to shrinking process including contact with water having less than boiling point / length of artificial fibroin fiber before performing the shrinking process)} × 100 (%)
[2]
The artificial fibroin fiber according to [1], wherein the modified fibroin is a modified spider silk fibroin.
[3]
The artificial fibroin fiber according to [1] or [2], wherein the temperature of the water is 10 to 90 ° C.
[4]
The artificial fibroin fiber according to any one of [1] to [3], wherein the shrinking process includes drying after contact with the water.
　本発明によれば、充分に高い収縮率を有し、かつ安全に製造が可能な人造フィブロイン繊維の提供が可能となる。 According to the present invention, it is possible to provide an artificial fibroin fiber that has a sufficiently high shrinkage rate and can be manufactured safely.
改変フィブロインのドメイン配列の一例を示す模式図である。It is a schematic diagram which shows an example of the domain arrangement | sequence of a modified fibroin. 天然由来のフィブロインのｚ／ｗ（％）の値の分布を示す図である。It is a figure which shows distribution of the value of z / w (%) of the fibroin derived from nature. 天然由来のフィブロインのｘ／ｙ（％）の値の分布を示す図である。It is a figure which shows distribution of the value of x / y (%) of naturally derived fibroin. 改変フィブロインのドメイン配列の一例を示す模式図である。It is a schematic diagram which shows an example of the domain arrangement | sequence of a modified fibroin. 人造フィブロイン繊維を製造するための紡糸装置の一例を概略的に示す説明図である。It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing artificial fibroin fiber. 人造フィブロイン繊維を収縮加工するための装置の一例を概略的に示す説明図である。It is explanatory drawing which shows roughly an example of the apparatus for shrink-processing an artificial fibroin fiber. 人造フィブロイン繊維を収縮加工するための装置の一例を概略的に示す説明図である。It is explanatory drawing which shows roughly an example of the apparatus for shrink-processing an artificial fibroin fiber.
　以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
〔人造フィブロイン繊維〕
　本発明に係る人造フィブロイン繊維は、改変フィブロインを含み、かつ水分（水や水蒸気等）と接触させるだけで高収縮が可能なものである。高収縮が可能とは、下記式で定義される収縮率が７％を超えることを意味する。
　収縮率＝｛１－（沸点未満の水に接触させることを含む収縮加工を施した人造フィブロイン繊維の長さ／収縮加工を施す前の人造フィブロイン繊維の長さ）｝×１００（％） [Artificial fibroin fiber]
The artificial fibroin fiber according to the present invention contains a modified fibroin and can be highly contracted only by contacting with moisture (water, water vapor, etc.). “High shrinkage is possible” means that the shrinkage rate defined by the following formula exceeds 7%.
Shrinkage rate = {1− (length of artificial fibroin fiber subjected to shrinkage processing including contact with water having less than boiling point / length of artificial fibroin fiber before being subjected to shrinkage processing)} × 100 (%)
＜改変フィブロイン＞
　本実施形態に係る改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。改変フィブロインは、ドメイン配列のＮ末端側及びＣ末端側のいずれか一方又は両方に更にアミノ酸配列（Ｎ末端配列及びＣ末端配列）が付加されていてもよい。Ｎ末端配列及びＣ末端配列は、これに限定されるものではないが、典型的には、フィブロインに特徴的なアミノ酸モチーフの反復を有さない領域であり、１００残基程度のアミノ酸からなる。 <Modified fibroin>
The modified fibroin according to the present embodiment has a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing. In the modified fibroin, an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to these, but are typically regions having no amino acid motif repeat characteristic of fibroin and consisting of about 100 amino acids.
　本明細書において「改変フィブロイン」とは、人為的に製造されたフィブロイン（人造フィブロイン）を意味する。改変フィブロインは、そのドメイン配列が、天然由来のフィブロインのアミノ酸配列とは異なるフィブロインであってもよく、天然由来のフィブロインとアミノ酸配列と同一であるフィブロインであってもよい。本明細書でいう「天然由来のフィブロイン」もまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。 As used herein, “modified fibroin” means an artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be a fibroin having the same amino acid sequence as that of naturally occurring fibroin. “Natural fibroin” as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. A protein comprising a domain sequence to be processed.
　「改変フィブロイン」は、本発明で特定されるアミノ酸配列を有するものであれば、天然由来のフィブロインのアミノ酸配列をそのまま利用したものであってもよく、天然由来のフィブロインのアミノ酸配列に依拠してそのアミノ酸配列を改変したもの（例えば、クローニングした天然由来のフィブロインの遺伝子配列を改変することによりアミノ酸配列を改変したもの）であってもよく、また天然由来のフィブロインに依らず人工的に設計及び合成したもの（例えば、設計したアミノ酸配列をコードする核酸を化学合成することにより所望のアミノ酸配列を有するもの）であってもよい。 As long as the “modified fibroin” has the amino acid sequence specified in the present invention, the amino acid sequence of naturally-occurring fibroin may be used as it is, depending on the amino acid sequence of naturally-occurring fibroin. The amino acid sequence may be modified (for example, the amino acid sequence may be modified by modifying the gene sequence of the cloned natural fibroin), and may be artificially designed and used independently of the natural fibroin. It may be synthesized (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).
　本明細書において「ドメイン配列」とは、フィブロイン特有の結晶領域（典型的には、アミノ酸配列の（Ａ）ｎモチーフに相当する。）と非晶領域（典型的には、アミノ酸配列のＲＥＰに相当する。）を生じるアミノ酸配列であり、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるアミノ酸配列を意味する。ここで、（Ａ）ｎモチーフは、アラニン残基を主とするアミノ酸配列を示し、アミノ酸残基数は２～２７である。（Ａ）ｎモチーフのアミノ酸残基数は、２～２０、４～２７、４～２０、８～２０、１０～２０、４～１６、８～１６、又は１０～１６の整数であってよい。また、（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数の割合は４０％以上であればよく、６０％以上、７０％以上、８０％以上、８３％以上、８５％以上、８６％以上、９０％以上、９５％以上、又は１００％（アラニン残基のみで構成されることを意味する。）であってもよい。ドメイン配列中に複数存在する（Ａ）ｎモチーフは、少なくとも７つがアラニン残基のみで構成されてもよい。ＲＥＰは２～２００アミノ酸残基から構成されるアミノ酸配列を示す。ＲＥＰは、１０～２００アミノ酸残基から構成されるアミノ酸配列であってもよい。ｍは２～３００の整数を示し、１０～３００の整数であってもよい。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 In the present specification, the “domain sequence” refers to a fibroin-specific crystal region (typically corresponding to the (A) n motif in the amino acid sequence) and an amorphous region (typically in the REP of the amino acid sequence). Amino acid sequence that gives rise to the following formula: Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) amino acid represented by n motif Means an array. Here, (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27. (A) 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 . In addition, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) n motif may be 40% or more, such as 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). A plurality of (A) n motifs present in the domain sequence may be composed of at least seven alanine residues alone. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. Plural REPs may have the same amino acid sequence or different amino acid sequences.
　（Ａ）ｎモチーフは、（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８３％以上であるのが好ましく、８６％以上であることがより好ましく、９０％以上であることが更に好ましく、９５％以上であることが更により好ましく、１００％であること（アラニン残基のみで構成されることを意味する）が特に好ましい。ドメイン配列中に複数存在する（Ａ）ｎモチーフは、少なくとも７つがアラニン残基のみで構成されることが好ましい。アラニン残基のみで構成されるとは、（Ａ）ｎモチーフが、（Ａ）ｎ（Ａはアラニン残基を示し、ｎは２～２０の整数、好ましくは４～２０、より好ましくは４～１６の整数を示す。）で表されるアミノ酸配列を有することを意味する。 (A) n motif, (A) is preferably alanine residues is 83% or more to the total number of amino acid residues in the n motif, more preferably 86% or more, 90% or more Is more preferably 95% or more, particularly preferably 100% (meaning that it is composed of only alanine residues). It is preferable that at least seven of the (A) n motifs present in the domain sequence are composed of only alanine residues. Consists of only alanine residues means that (A) n motif is (A) n (A represents an alanine residue, n is an integer of 2 to 20, preferably 4 to 20, more preferably 4 to It represents an integer of 16).
　本実施形態に係る改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列に対し、例えば、１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行うことで得ることができる。アミノ酸残基の置換、欠失、挿入及び／又は付加は、部分特異的突然変異誘発法等の当業者に周知の方法により行うことができる。具体的には、Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．１０，６４８７（１９８２）、Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，１００，４４８（１９８３）等の文献に記載されている方法に準じて行うことができる。 The modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or a plurality of amino acid residues to the cloned gene sequence of naturally derived fibroin. It can be obtained by modifying the above. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
　天然由来のフィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質であり、具体的には、例えば、昆虫又はクモ類が産生するフィブロインが挙げられる。 Naturally-derived fibroin is a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Specific examples include fibroin produced by insects or spiders.
　昆虫が産生するフィブロインとしては、例えば、ボンビックス・モリ（Ｂｏｍｂｙｘ　ｍｏｒｉ）、クワコ（Ｂｏｍｂｙｘ　ｍａｎｄａｒｉｎａ）、天蚕（Ａｎｔｈｅｒａｅａ　ｙａｍａｍａｉ）、柞蚕（Ａｎｔｅｒａｅａ　ｐｅｒｎｙｉ）、楓蚕（Ｅｒｉｏｇｙｎａ　ｐｙｒｅｔｏｒｕｍ）、蓖蚕（Ｐｉｌｏｓａｍｉａ　Ｃｙｎｔｈｉａ　ｒｉｃｉｎｉ）、樗蚕（Ｓａｍｉａ　ｃｙｎｔｈｉａ）、栗虫（Ｃａｌｉｇｕｒａ　ｊａｐｏｎｉｃａ）、チュッサー蚕（Ａｎｔｈｅｒａｅａ　ｍｙｌｉｔｔａ）、ムガ蚕（Ａｎｔｈｅｒａｅａ　ａｓｓａｍａ）等のカイコが産生する絹タンパク質、スズメバチ（Ｖｅｓｐａ　ｓｉｍｉｌｌｉｍａ　ｘａｎｔｈｏｐｔｅｒａ）の幼虫が吐出するホーネットシルクタンパク質が挙げられる。 Examples of fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandaraina), Tengea (Antherea yamanai), 柞 蚕 (Antereaperyi), 楓 蚕 (Eriothyraminey) ), Silkworms (Samia cythia), chestnut worms (Caligula japonica), Chussa moth (Anthereaea mylitta), silkworms produced by silkworms such as Antheraea assamata, vespasam worms Examples include silk proteins.
　昆虫が産生するフィブロインのより具体的な例としては、例えば、カイコ・フィブロインＬ鎖（ＧｅｎＢａｎｋアクセッション番号Ｍ７６４３０（塩基配列）、ＡＡＡ２７８４０．１（アミノ酸配列））が挙げられる。 More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).
　クモ類が産生するフィブロインとしては、例えば、オニグモ、ニワオニグモ、アカオニグモ、アオオニグモ及びマメオニグモ等のオニグモ属（Ａｒａｎｅｕｓ属）に属するクモ、ヤマシロオニグモ、イエオニグモ、ドヨウオニグモ及びサツマノミダマシ等のヒメオニグモ属（Ｎｅｏｓｃｏｎａ属）に属するクモ、コオニグモモドキ等のコオニグモモドキ属（Ｐｒｏｎｕｓ属）に属するクモ、トリノフンダマシ及びオオトリノフンダマシ等のトリノフンダマシ属（Ｃｙｒｔａｒａｃｈｎｅ属）に属するクモ、トゲグモ及びチブサトゲグモ等のトゲグモ属（Ｇａｓｔｅｒａｃａｎｔｈａ属）に属するクモ、マメイタイセキグモ及びムツトゲイセキグモ等のイセキグモ属（Ｏｒｄｇａｒｉｕｓ属）に属するクモ、コガネグモ、コガタコガネグモ及びナガコガネグモ等のコガネグモ属（Ａｒｇｉｏｐｅ属）に属するクモ、キジロオヒキグモ等のオヒキグモ属（Ａｒａｃｈｎｕｒａ属）に属するクモ、ハツリグモ等のハツリグモ属（Ａｃｕｓｉｌａｓ属）に属するクモ、スズミグモ、キヌアミグモ及びハラビロスズミグモ等のスズミグモ属（Ｃｙｔｏｐｈｏｒａ属）に属するクモ、ゲホウグモ等のゲホウグモ属（Ｐｏｌｔｙｓ属）に属するクモ、ゴミグモ、ヨツデゴミグモ、マルゴミグモ及びカラスゴミグモ等のゴミグモ属（Ｃｙｃｌｏｓａ属）に属するクモ、及びヤマトカナエグモ等のカナエグモ属（Ｃｈｏｒｉｚｏｐｅｓ属）に属するクモが産生するスパイダーシルクタンパク質、並びにアシナガグモ、ヤサガタアシナガグモ、ハラビロアシダカグモ及びウロコアシナガグモ等のアシナガグモ属（Ｔｅｔｒａｇｎａｔｈａ属）に属するクモ、オオシロカネグモ、チュウガタシロカネグモ及びコシロカネグモ等のシロカネグモ属（Ｌｅｕｃａｕｇｅ属）に属するクモ、ジョロウグモ及びオオジョロウグモ等のジョロウグモ属（Ｎｅｐｈｉｌａ属）に属するクモ、キンヨウグモ等のアズミグモ属（Ｍｅｎｏｓｉｒａ属）に属するクモ、ヒメアシナガグモ等のヒメアシナガグモ属（Ｄｙｓｃｈｉｒｉｏｇｎａｔｈａ属）に属するクモ、クロゴケグモ、セアカゴケグモ、ハイイロゴケグモ及びジュウサンボシゴケグモ等のゴケグモ属（Ｌａｔｒｏｄｅｃｔｕｓ属）に属するクモ、及びユープロステノプス属（Ｅｕｐｒｏｓｔｈｅｎｏｐｓ属）に属するクモ等のアシナガグモ科（Ｔｅｔｒａｇｎａｔｈｉｄａｅ科）に属するクモが産生するスパイダーシルクタンパク質が挙げられる。スパイダーシルクタンパク質としては、例えば、ＭａＳｐ（ＭａＳｐ１及びＭａＳｐ２）、ＡＤＦ（ＡＤＦ３及びＡＤＦ４）等の牽引糸タンパク質、ＭｉＳｐ（ＭｉＳｐ１及びＭｉＳｐ２）等が挙げられる。 Fibroin produced by spiders includes, for example, spiders belonging to the genus spider (Araneus spp.) Such as the spider spider, the spider spider, the red spider spider, and the bean spider, the genus spiders of the genus Araneus, the spider spider spider, the spider spider genus e Spiders, spiders such as spiders, spiders belonging to the genus Spider, spiders belonging to the genus Pronos, spiders belonging to the genus Trinofunda, such as Torinofundamas (genus Cyrtarachne) Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Spider (Ordgarius genus), such as the spiders, the spiders, and the spiders belonging to the genus Ordgarius Spiders belonging to the genus Argiope, such as the genus Argiope, spiders belonging to the genus Arachnura, such as the white-tailed spider, spiders belonging to the genus Acusilas such as the common spider, the spider belonging to the genus Acusilas, and the genus Spider Spiders belonging to (genus Cytophora), spiders belonging to the genus Spider belonging to the genus Spider (genus Poltys) such as spiders, genus Spider, spiders belonging to the genus Spider belonging to the genus Cyclosa (genus Cyclosa), and spiders belonging to the genus Cyclosa (genus Cyclosa) Spider silk proteins produced by spiders belonging to the genus Chorizopes), and Asina, such as Asagaaga spider, Yasagata Asaga spider, Harabiro Ashida spider and Urokoa Asaga spider Spiders belonging to the genus Tetragnata, spiders belonging to the genus Spider genus (Leucage sp.) Such as the white spider spider and the white spider spider, the spider genus belonging to the spider spider genus (Nephila spp. Spiders belonging to the genus Azumigumi (Menosira), spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the common spider spider, the black spider spider, the genus Spider genus belonging to the genus Spider belonging to the genus (L) and the genus Spider belonging to the genus (L) Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops Examples include spider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.
　クモ類が産生するフィブロインのより具体的な例としては、例えば、ｆｉｂｒｏｉｎ－３（ａｄｆ－３）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１０（アミノ酸配列）、Ｕ４７８５５（塩基配列））、ｆｉｂｒｏｉｎ－４（ａｄｆ－４）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１１（アミノ酸配列）、Ｕ４７８５６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ０４５０４（アミノ酸配列）、Ｕ３７５２０（塩基配列））、ｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｌａｔｒｏｄｅｃｔｕｓ　ｈｅｓｐｅｒｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ６８８５６（アミノ酸配列）、ＥＦ５９５２４６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖａｔａ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＬ３２４７２（アミノ酸配列）、ＡＦ４４１２４５（塩基配列））、ｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＣＡＪ００４２８（アミノ酸配列）、ＡＪ９７３１５５（塩基配列））、及びｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　２［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ］（ＧｅｎＢａｎｋアクセッション番号ＣＡＭ３２２４９．１（アミノ酸配列）、ＡＭ４９０１６９（塩基配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５８９．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５９１．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ－ｌｉｋｅ　ｐｒｏｔｅｉｎ［Ｎｅｐｈｉｌｅｎｇｙｓ　ｃｒｕｅｎｔａｔａ］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ３７２７８．１（アミノ酸配列）等が挙げられる。 More specific examples of fibroin produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin- 4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidolin 1 [derived from Nephila clavipes (GenBank accession number AAC4, amino acid sequence A04) U37520 (base sequence)), major sample spidroin 1 [La rodectus hesperus origin] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Nephila clavata origin] (GenBank accession number AAL32447, amino acid sequence 45, amino acid sequence) major amploidate 1 [from Euprothenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampulative spirodin 2 (EuprosentropisGastrosps nk accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampilate silk protein 1 [Nephila clavies] (GenBank accession number AAC1458.11 (amino acid sequence)), minor ampulativesilk2 ] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplitude spiroin-like protein [Nephilegenes cruentata] (GenBank accession number ABR37278. 1 (amino acid sequence), and the like.
　天然由来のフィブロインのより具体的な例としては、更に、ＮＣＢＩ　ＧｅｎＢａｎｋに配列情報が登録されているフィブロインを挙げることができる。例えば、ＮＣＢＩ　ＧｅｎＢａｎｋに登録されている配列情報のうちＤＩＶＩＳＩＯＮとしてＩＮＶを含む配列の中から、ＤＥＦＩＮＩＴＩＯＮにｓｐｉｄｒｏｉｎ、ａｍｐｕｌｌａｔｅ、ｆｉｂｒｏｉｎ、「ｓｉｌｋ及びｐｏｌｙｐｅｐｔｉｄｅ」、又は「ｓｉｌｋ及びｐｒｏｔｅｉｎ」がキーワードとして記載されている配列、ＣＤＳから特定のｐｒｏｄｕｃｔの文字列、ＳＯＵＲＣＥからＴＩＳＳＵＥ　ＴＹＰＥに特定の文字列の記載された配列を抽出することにより確認することができる。 More specific examples of naturally derived fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, spidin, sample, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION from sequences including INV as DIVISION among the sequence information registered in NCBI GenBank. It can be confirmed by extracting a character string of a specific product from the sequence, CDS, and a sequence in which the specific character string is described from SOURCE to TISSUE TYPE.
　本実施形態に係る改変フィブロインは、改変絹フィブロイン（カイコが産生する絹タンパク質のアミノ酸配列を改変したもの）であってもよく、改変クモ糸フィブロイン（クモ類が産生するスパイダーシルクタンパク質のアミノ酸配列を改変したもの）であってもよい。 The modified fibroin according to the present embodiment may be a modified silk fibroin (a modified amino acid sequence of a silk protein produced by a silkworm), and a modified spider silk fibroin (an amino acid sequence of a spider silk protein produced by a spider). It may be modified).
　本実施形態に係る改変フィブロインの具体的な例として、グリシン残基の含有量が低減された改変フィブロイン（第１の実施形態に係る改変フィブロイン）、（Ａ）ｎモチーフの含有量が低減された改変フィブロイン（第２の実施形態に係る改変フィブロイン）、グリシン残基の含有量、及び（Ａ）ｎモチーフの含有量が低減された改変フィブロイン（第３の実施形態に係る改変フィブロイン）、局所的に疎水性指標の大きい領域を含むドメイン配列を有する改変フィブロイン（第４の実施形態に係る改変フィブロイン）が挙げられる。 As specific examples of the modified fibroin according to the present embodiment, the modified fibroin with a reduced content of glycine residues (modified fibroin according to the first embodiment), (A) the content of the n motif is reduced. Modified fibroin (modified fibroin according to the second embodiment), content of glycine residue, and (A) modified fibroin with reduced content of n motif (modified fibroin according to the third embodiment), local And modified fibroin having a domain sequence including a region having a large hydrophobicity index (modified fibroin according to the fourth embodiment).
　第１の実施形態に係る改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、グリシン残基の含有量が低減されたアミノ酸配列を有する。当該改変フィブロインは、天然由来のフィブロインと比較して、少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。 The modified fibroin according to the first embodiment has an amino acid sequence in which the content of a glycine residue is reduced as compared with a naturally occurring fibroin. It can be said that the modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with naturally occurring fibroin.
　第１の実施形態に係る改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中のＧＧＸ及びＧＰＧＸＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）から選ばれる少なくとも一つのモチーフ配列において、少なくとも１又は複数の当該モチーフ配列中の１つのグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものであってもよい。 The modified fibroin according to the first embodiment has at least a domain sequence selected from GGX and GPGXX in REP (where X represents an amino acid residue other than glycine) as compared to naturally occurring fibroin. One motif sequence may have an amino acid sequence corresponding to the substitution of one glycine residue in at least one or a plurality of the motif sequences with another amino acid residue.
　第１の実施形態に係る改変フィブロインは、上述のグリシン残基が別のアミノ酸残基に置換されたモチーフ配列の割合が、全モチーフ配列に対して、１０％以上であってもよい。 In the modified fibroin according to the first embodiment, the ratio of the motif sequence in which the above-described glycine residue is substituted with another amino acid residue may be 10% or more with respect to the entire motif sequence.
　第１の実施形態に係る改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、上記ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を除いた配列中の全ＲＥＰに含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を除いた配列中の総アミノ酸残基数をｗとしたときに、ｚ／ｗが３０％以上、４０％以上、５０％以上又は５０．９％以上であるアミノ酸配列を有するものであってもよい。 The modified fibroin according to the first embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and (A) n motif located on the most C-terminal side from the domain sequence. Z is the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the domain sequence to the C-terminal of the domain sequence. When the total number of amino acid residues in the sequence excluding the sequence from the domain sequence to the most C-terminal (A) n motif to the C-terminus of the domain sequence is w, z / w is It may have an amino acid sequence that is 30% or more, 40% or more, 50% or more, or 50.9% or more.
　第１の実施形態に係る改変フィブロインは、ＧＧＸモチーフの１つのグリシン残基を別のアミノ酸残基に置換することにより。ＸＧＸからなるアミノ酸配列の含有割合を高めたものであることが好ましい。第１の実施形態に係る改変フィブロインは、ドメイン配列中のＧＧＸからなるアミノ酸配列の含有割合が３０％以下であることが好ましく、２０％以下であることがより好ましく、１０％以下であることが更に好ましく、６％以下であることが更により好ましく、４％以下であることが更によりまた好ましく、２％以下であることが特に好ましい。ドメイン配列中のＧＧＸからなるアミノ酸配列の含有割合は、下記ＸＧＸからなるアミノ酸配列の含有割合（ｚ／ｗ）の算出方法と同様の方法で算出することができる。 The modified fibroin according to the first embodiment is obtained by substituting one glycine residue of the GGX motif with another amino acid residue. It is preferable that the content ratio of the amino acid sequence consisting of XGX is increased. In the modified fibroin according to the first embodiment, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, and preferably 10% or less. More preferably, it is still more preferably 6% or less, still more preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.
　ｚ／ｗの算出方法を更に詳細に説明する。まず、ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列に含まれる全てのＲＥＰから、ＸＧＸからなるアミノ酸配列を抽出する。ＸＧＸを構成するアミノ酸残基の総数がｚである。例えば、ＸＧＸからなるアミノ酸配列が５０個抽出された場合（重複はなし）、ｚは５０×３＝１５０である。また、例えば、ＸＧＸＧＸからなるアミノ酸配列の場合のように２つのＸＧＸに含まれるＸ（中央のＸ）が存在する場合は、重複分を控除して計算する（ＸＧＸＧＸの場合は５アミノ酸残基である）。ｗは、ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列に含まれる総アミノ酸残基数である。例えば、図１に示したドメイン配列の場合、ｗは４＋５０＋４＋１００＋４＋１０＋４＋２０＋４＋３０＝２３０である（最もＣ末端側に位置する（Ａ）ｎモチーフは除いている。）。次に、ｚをｗで除すことによって、ｚ／ｗ（％）を算出することができる。 The method for calculating z / w will be described in more detail. First, an amino acid sequence consisting of XGX is extracted from all REPs included in the sequence excluding the sequence from the domain motif (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50 × 3 = 150. Also, for example, when there is an X (center X) included in two XGXs as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, it is 5 amino acid residues). is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the domain sequence to the most C-terminal (A) n motif to the C-terminus of the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A) n motif located closest to the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.
　ここで、天然由来のフィブロインにおけるｚ／ｗについて説明する。まず、上述のように、ＮＣＢＩ　ＧｅｎＢａｎｋにアミノ酸配列情報が登録されているフィブロインを例示した方法により確認したところ、６６３種類のフィブロイン（このうち、クモ類由来のフィブロインは４１５種類）が抽出された。抽出された全てのフィブロインのうち、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、フィブロイン中のＧＧＸからなるアミノ酸配列の含有割合が６％以下である天然由来のフィブロインのアミノ酸配列から、上述の算出方法により、ｚ／ｗを算出した。その結果を図２に示す。図２の横軸はｚ／ｗ（％）を示し、縦軸は頻度を示す。図２から明らかなとおり、天然由来のフィブロインにおけるｚ／ｗは、いずれも５０．９％未満である（最も高いもので、５０．８６％）。 Here, z / w in naturally derived fibroin will be described. First, as described above, when a fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by a method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Of all the extracted fibroin, it is derived from the natural origin including the domain sequence represented by Formula 1: [(A) n motif-REP] m , and the content ratio of the amino acid sequence consisting of GGX in fibroin is 6% or less Z / w was calculated from the amino acid sequence of the fibroin by the calculation method described above. The result is shown in FIG. The horizontal axis in FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, z / w in naturally derived fibroin is less than 50.9% (the highest is 50.86%).
　第１の実施形態に係る改変フィブロインにおいて、ｚ／ｗは、５０．９％以上であることが好ましく、５６．１％以上であることがより好ましく、５８．７％以上であることが更に好ましく、７０％以上であることが更により好ましく、８０％以上であることが更によりまた好ましい。ｚ／ｗの上限に特に制限はないが、例えば、９５％以下であってもよい。 In the modified fibroin according to the first embodiment, z / w is preferably 50.9% or more, more preferably 56.1% or more, and further preferably 58.7% or more. 70% or more is even more preferable, and 80% or more is even more preferable. Although there is no restriction | limiting in particular in the upper limit of z / w, For example, 95% or less may be sufficient.
　第１の実施形態に係る改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列から、グリシン残基をコードする塩基配列の少なくとも一部を置換して別のアミノ酸残基をコードするように改変することにより得ることができる。このとき、改変するグリシン残基として、ＧＧＸモチーフ及びＧＰＧＸＸモチーフにおける１つのグリシン残基を選択してもよいし、またｚ／ｗが５０．９％以上になるように置換してもよい。また、例えば、天然由来のフィブロインのアミノ酸配列から上記態様を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列からＲＥＰ中のグリシン残基を別のアミノ酸残基に置換したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 In the modified fibroin according to the first embodiment, for example, from the cloned gene sequence of naturally-occurring fibroin, at least a part of the base sequence encoding a glycine residue is substituted to encode another amino acid residue. It can be obtained by modifying. At this time, one glycine residue in GGX motif and GPGXX motif may be selected as a glycine residue to be modified, or substitution may be performed so that z / w is 50.9% or more. In addition, for example, an amino acid sequence satisfying the above-described aspect can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis. In any case, in addition to the modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. The amino acid sequence corresponding to the insertion and / or addition may be modified.
　上記の別のアミノ酸残基としては、グリシン残基以外のアミノ酸残基であれば特に制限はないが、バリン（Ｖ）残基、ロイシン（Ｌ）残基、イソロイシン（Ｉ）残基、メチオニン（Ｍ）残基、プロリン（Ｐ）残基、フェニルアラニン（Ｆ）残基及びトリプトファン（Ｗ）残基等の疎水性アミノ酸残基、グルタミン（Ｑ）残基、アスパラギン（Ｎ）残基、セリン（Ｓ）残基、リシン（Ｋ）残基及びグルタミン酸（Ｅ）残基等の親水性アミノ酸残基が好ましく、バリン（Ｖ）残基、ロイシン（Ｌ）残基、イソロイシン（Ｉ）残基及びグルタミン（Ｑ）残基がより好ましく、グルタミン（Ｑ）残基が更に好ましい。 The other amino acid residue is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( M) hydrophobic amino acid residues such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and glutamic acid (E) residues are preferred, and valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residue is more preferable, and glutamine (Q) residue is more preferable.
　第１の実施形態に係る改変フィブロインのより具体的な例として、（１－ｉ）配列番号３、配列番号４、配列番号１０若しくは配列番号１２で示されるアミノ酸配列、又は（１－ｉｉ）配列番号３、配列番号４、配列番号１０若しくは配列番号１２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of the modified fibroin according to the first embodiment, (1-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (1-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more of sequence identity with the amino acid sequence shown in No. 3, SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12.
　（１－ｉ）の改変フィブロインについて説明する。配列番号３で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１で示されるアミノ酸配列のＲＥＰ中の全てのＧＧＸをＧＱＸに置換したものである。配列番号４で示されるアミノ酸配列は、配列番号３で示されるアミノ酸配列から、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフを欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したものである。配列番号１０で示されるアミノ酸配列は、配列番号４で示されるアミノ酸配列の各（Ａ）ｎモチーフのＣ末端側に２つのアラニン残基を挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、配列番号４の分子量とほぼ同じとなるようにＮ末端側の一部のアミノ酸を欠失させたものである。配列番号１２で示されるアミノ酸配列は、配列番号９で示されるアミノ酸配列中に存在する２０個のドメイン配列の領域（但し、当該領域のＣ末端側の数アミノ酸残基が置換されている。）を４回繰り返した配列のＣ末端にＨｉｓタグが付加されたものである。 The modified fibroin (1-i) will be described. The amino acid sequence represented by SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin. The amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence represented by SEQ ID NO: 3, in which every two (A) n motifs are deleted from the N-terminal side to the C-terminal side, and further before the C-terminal sequence. One [(A) n motif-REP] is inserted into the. The amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
　配列番号１で示されるアミノ酸配列（天然由来のフィブロインに相当）におけるｚ／ｗの値は、４６．８％である。配列番号３で示されるアミノ酸配列、配列番号４で示されるアミノ酸配列、配列番号１０で示されるアミノ酸配列、及び配列番号１２で示されるアミノ酸配列におけるｚ／ｗの値は、それぞれ５８．７％、７０．１％、６６．１％及び７０．０％である。また、配列番号１、配列番号３、配列番号４、配列番号１０及び配列番号１２で示されるアミノ酸配列のギザ比率（後述する）１：１．８～１１．３におけるｘ／ｙの値は、それぞれ１５．０％、１５．０％、９３．４％、９２．７％及び８９．３％である。 The value of z / w in the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 12 are 58.7%, 70.1%, 66.1% and 70.0%. In addition, the value of x / y at the ratio of the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 (described later) 1: 1.8 to 11.3 is: 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.
　（１－ｉ）の改変フィブロインは、配列番号３、配列番号４、配列番号１０又は配列番号１２で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin (1-i) may consist of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
　（１－ｉｉ）の改変フィブロインは、配列番号３、配列番号４、配列番号１０又は配列番号１２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（１－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin (1-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (1-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（１－ｉｉ）の改変フィブロインは、配列番号３、配列番号４、配列番号１０又は配列番号１２で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＲＥＰ中に含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列中のＲＥＰの総アミノ酸残基数をｗとしたときに、ｚ／ｗが５０．９％以上であることが好ましい。 The modified fibroin of (1-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
　上述の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、改変フィブロインの単離、固定化、検出及び可視化等が可能となる。 The above-mentioned modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
　タグ配列として、例えば、他の分子との特異的親和性（結合性、アフィニティ）を利用したアフィニティタグを挙げることができる。アフィニティタグの具体例として、ヒスチジンタグ（Ｈｉｓタグ）を挙げることができる。Ｈｉｓタグは、ヒスチジン残基が４から１０個程度並んだ短いペプチドで、ニッケル等の金属イオンと特異的に結合する性質があるため、金属キレートクロマトグラフィー（ｃｈｅｌａｔｉｎｇ　ｍｅｔａｌ　ｃｈｒｏｍａｔｏｇｒａｐｈｙ）による改変フィブロインの単離に利用することができる。タグ配列の具体例として、例えば、配列番号５で示されるアミノ酸配列（Ｈｉｓタグを含むアミノ酸配列）が挙げられる。 Examples of tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules. Specific examples of the affinity tag include a histidine tag (His tag). The His tag is a short peptide with about 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography) Can be used. Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 5 (amino acid sequence containing a His tag).
　また、グルタチオンに特異的に結合するグルタチオン－Ｓ－トランスフェラーゼ（ＧＳＴ）、マルトースに特異的に結合するマルトース結合タンパク質（ＭＢＰ）等のタグ配列を利用することもできる。 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.
　さらに、抗原抗体反応を利用した「エピトープタグ」を利用することもできる。抗原性を示すペプチド（エピトープ）をタグ配列として付加することにより、当該エピトープに対する抗体を結合させることができる。エピトープタグとして、ＨＡ（インフルエンザウイルスのヘマグルチニンのペプチド配列）タグ、ｍｙｃタグ、ＦＬＡＧタグ等を挙げることができる。エピトープタグを利用することにより、高い特異性で容易に改変フィブロインを精製することができる。 Furthermore, an “epitope tag” using an antigen-antibody reaction can also be used. By adding a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound. As the epitope tag, HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like can be mentioned. By using the epitope tag, the modified fibroin can be easily purified with high specificity.
　さらにタグ配列を特定のプロテアーゼで切り離せるようにしたものも使用することができる。当該タグ配列を介して吸着したタンパク質をプロテアーゼ処理することにより、タグ配列を切り離した改変フィブロインを回収することもできる。 Furthermore, a tag sequence that can be separated with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with protease, the modified fibroin from which the tag sequence has been separated can also be recovered.
　タグ配列を含む改変フィブロインのより具体的な例として、（１－ｉｉｉ）配列番号８、配列番号９、配列番号１１若しく配列番号１３で示されるアミノ酸配列、又は（１－ｉｖ）配列番号８、配列番号９、配列番号１１若しく配列番号１３で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 More specific examples of the modified fibroin containing the tag sequence include (1-iii) the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (1-iv) SEQ ID NO: 8 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
　配列番号６、配列番号７、配列番号８、配列番号９、配列番号１１及び配列番号１３で示されるアミノ酸配列は、それぞれ配列番号１、配列番号２、配列番号３、配列番号４、配列番号１０及び配列番号１２で示されるアミノ酸配列のＮ末端に配列番号５で示されるアミノ酸配列（Ｈｉｓタグを含む）を付加したものである。 The amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively. And the amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
　（１－ｉｉｉ）の改変フィブロインは、配列番号８、配列番号９、配列番号１１又は配列番号１３で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin (1-iii) may be composed of the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
　（１－ｉｖ）の改変フィブロインは、配列番号８、配列番号９、配列番号１１又は配列番号１３で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（１－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin (1-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (1-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（１－ｉｖ）の改変フィブロインは、配列番号８、配列番号９、配列番号１１又は配列番号１３で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＲＥＰ中に含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列中のＲＥＰの総アミノ酸残基数をｗとしたときに、ｚ／ｗが５０．９％以上であることが好ましい。 The modified fibroin (1-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
　上述の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 The aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of host.
　第２の実施形態に係る改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、（Ａ）ｎモチーフの含有量が低減されたアミノ酸配列を有する。当該改変フィブロインのドメイン配列は、天然由来のフィブロインと比較して、少なくとも１又は複数の（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものということができる。 The modified fibroin according to the second embodiment has an amino acid sequence in which the domain sequence has a reduced content of (A) n motif compared to naturally occurring fibroin. It can be said that the domain sequence of the modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) n motifs as compared to naturally occurring fibroin.
　第２の実施形態に係る改変フィブロインは、天然由来のフィブロインから（Ａ）ｎモチーフを１０～４０％欠失させたことに相当するアミノ酸配列を有するものであってもよい。 The modified fibroin according to the second embodiment may have an amino acid sequence corresponding to 10% to 40% deletion of the (A) n motif from naturally occurring fibroin.
　第２の実施形態に係る改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、少なくともＮ末端側からＣ末端側に向かって１～３つの（Ａ）ｎモチーフ毎に１つの（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。 The modified fibroin according to the second embodiment has a domain sequence of 1 to 3 (A) n motifs at least from the N-terminal side to the C-terminal side as compared with naturally-occurring fibroin ( A) It may have an amino acid sequence corresponding to deletion of the n motif.
　第２の実施形態に係る改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、少なくともＮ末端側からＣ末端側に向かって２つ連続した（Ａ）ｎモチーフの欠失、及び１つの（Ａ）ｎモチーフの欠失がこの順に繰り返されたことに相当するアミノ酸配列を有するものであってもよい。 The modified fibroin according to the second embodiment has a domain sequence that is at least two consecutive from the N-terminal side to the C-terminal side compared to naturally-occurring fibroin (A) deletion of the n motif, and It may have an amino acid sequence corresponding to the deletion of one (A) n motif repeated in this order.
　第２の実施形態に係る改変フィブロインは、そのドメイン配列が、少なくともＮ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。 The modified fibroin according to the second embodiment has an amino acid sequence corresponding to the deletion of every two (A) n motifs at least every two domain sequences from the N-terminal side to the C-terminal side. There may be.
　第２の実施形態に係る改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、Ｎ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが２０％以上、３０％以上、４０％以上又は５０％以上であるアミノ酸配列を有するものであってもよい。 The modified fibroin according to the second embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [[( A) The number of amino acid residues in the REP of the n motif-REP] unit is sequentially compared, and when the number of amino acid residues in the REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is The maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units that are 1.8 to 11.3 is x, and the total number of amino acid residues in the domain sequence is When y, x / y may have an amino acid sequence of 20% or more, 30% or more, 40% or more, or 50% or more.
　ｘ／ｙの算出方法を図１を参照しながら更に詳細に説明する。図１には、改変フィブロインからＮ末端配列及びＣ末端配列を除いたドメイン配列を示す。当該ドメイン配列は、Ｎ末端側（左側）から（Ａ）ｎモチーフ－第１のＲＥＰ（５０アミノ酸残基）－（Ａ）ｎモチーフ－第２のＲＥＰ（１００アミノ酸残基）－（Ａ）ｎモチーフ－第３のＲＥＰ（１０アミノ酸残基）－（Ａ）ｎモチーフ－第４のＲＥＰ（２０アミノ酸残基）－（Ａ）ｎモチーフ－第５のＲＥＰ（３０アミノ酸残基）－（Ａ）ｎモチーフという配列を有する。 The method for calculating x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. The domain sequence is from the N-terminal side (left side): (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called n motif.
　隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットは、重複がないように、Ｎ末端側からＣ末端側に向かって、順次選択する。このとき、選択されない［（Ａ）ｎモチーフ－ＲＥＰ］ユニットが存在してもよい。図１には、パターン１（第１のＲＥＰと第２のＲＥＰの比較、及び第３のＲＥＰと第４のＲＥＰの比較）、パターン２（第１のＲＥＰと第２のＲＥＰの比較、及び第４のＲＥＰと第５のＲＥＰの比較）、パターン３（第２のＲＥＰと第３のＲＥＰの比較、及び第４のＲＥＰと第５のＲＥＰの比較）、パターン４（第１のＲＥＰと第２のＲＥＰの比較）を示した。なお、これ以外にも選択方法は存在する。 Two adjacent [(A) n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so as not to overlap. At this time, an unselected [(A) n motif-REP] unit may exist. FIG. 1 includes pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison), pattern 4 (first REP and Comparison of the second REP). There are other selection methods.
　次に各パターンについて、選択した隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニット中の各ＲＥＰのアミノ酸残基数を比較する。比較は、よりアミノ酸残基数の少ない方を１としたときの、他方のアミノ酸残基数の比を求めることによって行う。例えば、第１のＲＥＰ（５０アミノ酸残基）と第２のＲＥＰ（１００アミノ酸残基）の比較の場合、よりアミノ酸残基数の少ない第１のＲＥＰを１としたとき、第２のＲＥＰのアミノ酸残基数の比は、１００／５０＝２である。同様に、第４のＲＥＰ（２０アミノ酸残基）と第５のＲＥＰ（３０アミノ酸残基）の比較の場合、よりアミノ酸残基数の少ない第４のＲＥＰを１としたとき、第５のＲＥＰのアミノ酸残基数の比は、３０／２０＝１．５である。 Next, for each pattern, the number of amino acid residues of each REP in the two adjacent [(A) n motif-REP] units selected is compared. The comparison is performed by determining the ratio of the number of amino acid residues on the other side when the smaller number of amino acid residues is 1. For example, in the comparison of the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the comparison of the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is 1, the fifth REP The ratio of the number of amino acid residues is 30/20 = 1.5.
　図１中、よりアミノ酸残基数の少ない方を１としたときに、他方のアミノ酸残基数の比が１．８～１１．３となる［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの組を実線で示した。以下このような比をギザ比率と呼ぶ。よりアミノ酸残基数の少ない方を１としたときに、他方のアミノ酸残基数の比が１．８未満又は１１．３超となる［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの組は破線で示した。 In FIG. 1, when the smaller number of amino acid residues is 1, the ratio of the number of other amino acid residues is 1.8 to 11.3 [(A) n motif-REP] unit pairs Shown in solid line. Hereinafter, such a ratio is referred to as a jagged ratio. When the smaller number of amino acid residues is 1, the ratio of the number of other amino acid residues is less than 1.8 or more than 11.3. [(A) n motif-REP] unit pairs are indicated by broken lines. Indicated.
　各パターンにおいて、実線で示した隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの全てのアミノ酸残基数を足し合わせる（ＲＥＰのみではなく、（Ａ）ｎモチーフのアミノ酸残基数もである。）。そして、足し合わせた合計値を比較して、当該合計値が最大となるパターンの合計値（合計値の最大値）をｘとする。図１に示した例では、パターン１の合計値が最大である。 In each pattern, the number of all amino acid residues of two adjacent [(A) n motif-REP] units indicated by solid lines is added (not only REP but also (A) the number of amino acid residues of the n motif. is there.). Then, the total value added is compared, and the total value (maximum value of the total value) of the pattern having the maximum total value is set as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
　次に、ｘをドメイン配列の総アミノ酸残基数ｙで除すことによって、ｘ／ｙ（％）を算出することができる。 Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y of the domain sequence.
　第２の実施形態に係る改変フィブロインにおいて、ｘ／ｙは、５０％以上であることが好ましく、６０％以上であることがより好ましく、６５％以上であることが更に好ましく、７０％以上であることが更により好ましく、７５％以上であることが更によりまた好ましく、８０％以上であることが特に好ましい。ｘ／ｙの上限に特に制限はなく、例えば、１００％以下であってよい。ギザ比率が１：１．９～１１．３の場合には、ｘ／ｙは８９．６％以上であることが好ましく、ギザ比率が１：１．８～３．４の場合には、ｘ／ｙは７７．１％以上であることが好ましく、ギザ比率が１：１．９～８．４の場合には、ｘ／ｙは７５．９％以上であることが好ましく、ギザ比率が１：１．９～４．１の場合には、ｘ／ｙは６４．２％以上であることが好ましい。 In the modified fibroin according to the second embodiment, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, and 70% or more. Is more preferably 75% or more, still more preferably 80% or more. There is no restriction | limiting in particular in the upper limit of x / y, For example, you may be 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x / y / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.
　第２の実施形態に係る改変フィブロインが、ドメイン配列中に複数存在する（Ａ）ｎモチーフの少なくとも７つがアラニン残基のみで構成される改変フィブロインである場合、ｘ／ｙは、４６．４％以上であることが好ましく、５０％以上であることがより好ましく、５５％以上であることが更に好ましく、６０％以上であることが更により好ましく、７０％以上であることが更によりまた好ましく、８０％以上であることが特に好ましい。ｘ／ｙの上限に特に制限はなく、１００％以下であればよい。 When the modified fibroin according to the second embodiment is plural in the domain sequence (A) When at least 7 of the n motifs are modified fibroin composed of only alanine residues, x / y is 46.4% Preferably, it is 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, It is especially preferable that it is 80% or more. There is no restriction | limiting in particular in the upper limit of x / y, and what is necessary is just 100% or less.
　ここで、天然由来のフィブロインにおけるｘ／ｙについて説明する。まず、上述のように、ＮＣＢＩ　ＧｅｎＢａｎｋにアミノ酸配列情報が登録されているフィブロインを例示した方法により確認したところ、６６３種類のフィブロイン（このうち、クモ類由来のフィブロインは４１５種類）が抽出された。抽出された全てのフィブロインのうち、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列で構成される天然由来のフィブロインのアミノ酸配列から、上述の算出方法により、ｘ／ｙを算出した。ギザ比率が１：１．９～４．１の場合の結果を図３に示す。 Here, x / y in naturally derived fibroin will be described. First, as described above, when a fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by a method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Of all the extracted fibroin, x / y is calculated from the amino acid sequence of naturally derived fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above-described calculation method. Was calculated. FIG. 3 shows the results when the jagged ratio is 1: 1.9 to 4.1.
　図３の横軸はｘ／ｙ（％）を示し、縦軸は頻度を示す。図３から明らかなとおり、天然由来のフィブロインにおけるｘ／ｙは、いずれも６４．２％未満である（最も高いもので、６４．１４％）。 3, the horizontal axis indicates x / y (%), and the vertical axis indicates frequency. As is clear from FIG. 3, x / y in naturally derived fibroin is less than 64.2% (the highest, 64.14%).
　第２の実施形態に係る改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列から、ｘ／ｙが６４．２％以上になるように（Ａ）ｎモチーフをコードする配列の１又は複数を欠失させることにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から、ｘ／ｙが６４．２％以上になるように１又は複数の（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列から（Ａ）ｎモチーフが欠失したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 The modified fibroin according to the second embodiment includes, for example, one or a plurality of sequences encoding the n motif so that x / y is 64.2% or more from the cloned gene sequence of natural fibroin (A) Can be obtained by deleting. In addition, for example, an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. In any case, in addition to the modification corresponding to the deletion of the (A) n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. The amino acid sequence corresponding to this may be modified.
　第２の実施形態に係る改変フィブロインのより具体的な例として、（２－ｉ）配列番号２、配列番号４、配列番号１０若しくは配列番号１２で示されるアミノ酸配列、又は（２－ｉｉ）配列番号２、配列番号４、配列番号１０若しくは配列番号１２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As a more specific example of the modified fibroin according to the second embodiment, (2-i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in No. 2, SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12.
　（２－ｉ）の改変フィブロインについて説明する。配列番号２で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１で示されるアミノ酸配列から、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフを欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したものである。配列番号４で示されるアミノ酸配列は、配列番号２で示されるアミノ酸配列のＲＥＰ中の全てのＧＧＸをＧＱＸに置換したものである。配列番号１０で示されるアミノ酸配列は、配列番号４で示されるアミノ酸配列の各（Ａ）ｎモチーフのＣ末端側に２つのアラニン残基を挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、配列番号４の分子量とほぼ同じとなるようにＮ末端側の一部のアミノ酸を欠失させたものである。配列番号１２で示されるアミノ酸配列は、配列番号９で示されるアミノ酸配列中に存在する２０個のドメイン配列の領域（但し、当該領域のＣ末端側の数アミノ酸残基が置換されている。）を４回繰り返した配列のＣ末端にＨｉｓタグが付加されたものである。 The modified fibroin (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 2 has the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin deleted from the N-terminal side to the C-terminal side every two (A) n motifs Furthermore, one [(A) n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting all GGX in REP of the amino acid sequence shown in SEQ ID NO: 2 with GQX. The amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
　配列番号１で示されるアミノ酸配列（天然由来のフィブロインに相当）のギザ比率１：１．８～１１．３におけるｘ／ｙの値は１５．０％である。配列番号２で示されるアミノ酸配列、及び配列番号４で示されるアミノ酸配列におけるｘ／ｙの値は、いずれも９３．４％である。配列番号１０で示されるアミノ酸配列におけるｘ／ｙの値は、９２．７％である。配列番号１２で示されるアミノ酸配列におけるｘ／ｙの値は、８９．８％である。配列番号１、配列番号２、配列番号４、配列番号１０及び配列番号１２で示されるアミノ酸配列におけるｚ／ｗの値は、それぞれ４６．８％、５６．２％、７０．１％、６６．１％及び８０．４％である。 The value of x / y of the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally-occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 4 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 12 is 89.8%. The z / w values in the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 are 46.8%, 56.2%, 70.1% and 66. respectively. 1% and 80.4%.
　（２－ｉ）の改変フィブロインは、配列番号２、配列番号４、配列番号１０又は配列番号１２で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin (2-i) may be composed of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
　（２－ｉｉ）の改変フィブロインは、配列番号２、配列番号４、配列番号１０又は配列番号１２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（２－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（２－ｉｉ）の改変フィブロインは、配列番号２、配列番号４、配列番号１０又は配列番号１２で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＮ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３（ギザ比率が１：１．８～１１．３）となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが６４．２％以上であることが好ましい。 The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and from the N-terminal side to the C-terminal side When the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other The amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio is 1: 1.8 to 11.3) It is preferable that x / y is 64.2% or more, where x is the maximum total value of the total number of bases and y is the total number of amino acid residues in the domain sequence.
　上述の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方に上述したタグ配列を含んでいてもよい。 The above-described modified fibroin may contain the above-described tag sequence at one or both of the N-terminal and C-terminal.
　タグ配列を含む改変フィブロインのより具体的な例として、（２－ｉｉｉ）配列番号７、配列番号９、配列番号１１若しく配列番号１３で示されるアミノ酸配列、又は（２－ｉｖ）配列番号７、配列番号９、配列番号１１若しく配列番号１３で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 More specific examples of the modified fibroin containing the tag sequence include (2-iii) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 7 And a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
　配列番号６、配列番号７、配列番号８、配列番号９、配列番号１１及び配列番号１３で示されるアミノ酸配列は、それぞれ配列番号１、配列番号２、配列番号３、配列番号４、配列番号１０及び配列番号１２で示されるアミノ酸配列のＮ末端に配列番号５で示されるアミノ酸配列（Ｈｉｓタグを含む）を付加したものである。 The amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively. And the amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
　（２－ｉｉｉ）の改変フィブロインは、配列番号７、配列番号９、配列番号１１又は配列番号１３で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin (2-iii) may be composed of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
　（２－ｉｖ）の改変フィブロインは、配列番号７、配列番号９、配列番号１１又は配列番号１３で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（２－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（２－ｉｖ）の改変フィブロインは、配列番号７、配列番号９、配列番号１１又は配列番号１３で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＮ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが６４．２％以上であることが好ましい。 The modified fibroin (2-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and from the N-terminal side to the C-terminal side. When the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other X is the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. When the total number of amino acid residues of the domain sequence is y, x / y is preferably 64.2% or more.
　上述の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 The aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of host.
　第３の実施形態に係る改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、（Ａ）ｎモチーフの含有量が低減されたことに加え、グリシン残基の含有量が低減されたアミノ酸配列を有するものである。当該改変フィブロインのドメイン配列は、天然由来のフィブロインと比較して、少なくとも１又は複数の（Ａ）ｎモチーフが欠失したことに加え、更に少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。すなわち、上述した第１の実施形態に係る改変フィブロインと、第２の実施形態に係る改変フィブロインの特徴を併せ持つ改変フィブロインである。具体的な態様等は、第１及び第２の実施形態に係る改変フィブロインで説明したとおりである。 In the modified fibroin according to the third embodiment, the domain sequence has a reduced content of glycine residues in addition to the reduced content of the (A) n motif compared to the naturally derived fibroin. Have an amino acid sequence. In addition to the deletion of at least one or more (A) n motifs, the domain sequence of the modified fibroin is different from that of naturally occurring fibroin in addition to at least one or more glycine residues in REP. It can be said to have an amino acid sequence corresponding to substitution with an amino acid residue. That is, the modified fibroin having the characteristics of the modified fibroin according to the first embodiment described above and the modified fibroin according to the second embodiment. Specific aspects and the like are as described in the modified fibroin according to the first and second embodiments.
　第３の実施形態に係る改変フィブロインのより具体的な例として、（３－ｉ）配列番号４、配列番号１０若しくは配列番号１２で示されるアミノ酸配列、（３－ｉｉ）配列番号４、配列番号１０若しくは配列番号１２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。配列番号４、配列番号１０若しくは配列番号１２で示されるアミノ酸配列を含む改変フィブロインの具体的な態様は上述のとおりである。 More specific examples of the modified fibroin according to the third embodiment include (3-i) an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, (3-ii) SEQ ID NO: 4, SEQ ID NO: Examples thereof include modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 10 or SEQ ID NO: 12. Specific embodiments of the modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.
　第４の実施形態に係る改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する、局所的に疎水性指標の大きい領域を含むアミノ酸配列を有するものであってよい。 The modified fibroin according to the fourth embodiment has a domain sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues having a large hydrophobicity index as compared with naturally occurring fibroin, And / or having an amino acid sequence that locally includes a region having a large hydrophobicity index corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index in REP.
　局所的に疎水性指標の大きい領域は、連続する２～４アミノ酸残基で構成されていることが好ましい。 The region where the hydrophobic index is locally large is preferably composed of 2 to 4 amino acid residues.
　上述の疎水性指標の大きいアミノ酸残基は、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）から選ばれるアミノ酸残基であることがより好ましい。 The amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). More preferably, it is a residue.
　第４の実施形態に係る改変フィブロインは、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に、天然由来のフィブロインと比較して、１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変があってもよい。 The modified fibroin according to the fourth embodiment has one or more amino acid residues in REP substituted with amino acid residues having a large hydrophobicity index and / or in REP compared to naturally occurring fibroin. In addition to the modification corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index, the substitution, deletion or insertion of one or more amino acid residues as compared with naturally occurring fibroin And / or there may be amino acid sequence modifications corresponding to the addition.
　第４の実施形態に係る改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列からＲＥＰ中の１又は複数の親水性アミノ酸残基（例えば、疎水性指標がマイナスであるアミノ酸残基）を疎水性アミノ酸残基（例えば、疎水性指標がプラスであるアミノ酸残基）に置換すること、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 The modified fibroin according to the fourth embodiment includes, for example, one or more hydrophilic amino acid residues (for example, amino acid residues whose hydrophobicity index is negative) in REP from the cloned gene sequence of naturally-occurring fibroin. It can be obtained by substituting with a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. In addition, for example, one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to insertion of, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more hydrophilic amino acid residues in REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin and / or one or more hydrophobic amino acids in REP In addition to the modification corresponding to the insertion of a residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.
　第４の実施形態に係る改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を上記ドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を上記ドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であるアミノ酸配列を有してもよい。 The modified fibroin according to the fourth embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side (A) from the n motif of the domain sequence. The total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence obtained by removing the sequence up to the C-terminal from the domain sequence. P, and (A) where the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is q / Q may have an amino acid sequence of 6.2% or more.
　アミノ酸残基の疎水性指標については、公知の指標（Ｈｙｄｒｏｐａｔｈｙ　ｉｎｄｅｘ：Ｋｙｔｅ　Ｊ，＆Ｄｏｏｌｉｔｔｌｅ　Ｒ（１９８２）“Ａ　ｓｉｍｐｌｅ　ｍｅｔｈｏｄ　ｆｏｒ　ｄｉｓｐｌａｙｉｎｇ　ｔｈｅ　ｈｙｄｒｏｐａｔｈｉｃ　ｃｈａｒａｃｔｅｒ　ｏｆ　ａ　ｐｒｏｔｅｉｎ”，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１５７，ｐｐ．１０５－１３２）を使用する。具体的には、各アミノ酸の疎水性指標（ハイドロパシー・インデックス、以下「ＨＩ」とも記す。）は、下記表１に示すとおりである。 As for the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of bio.p. 7”. 105-132). Specifically, the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　ｐ／ｑの算出方法を更に詳細に説明する。算出には、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列（以下、「配列Ａ」とする）を用いる。まず、配列Ａに含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値を算出する。疎水性指標の平均値は、連続する４アミノ酸残基に含まれる各アミノ酸残基のＨＩの総和を４（アミノ酸残基数）で除して求める。疎水性指標の平均値は、全ての連続する４アミノ酸残基について求める（各アミノ酸残基は、１～４回平均値の算出に用いられる。）。次いで、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域を特定する。あるアミノ酸残基が、複数の「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」に該当する場合であっても、領域中には１アミノ酸残基として含まれることになる。そして、当該領域に含まれるアミノ酸残基の総数がｐである。また、配列Ａに含まれるアミノ酸残基の総数がｑである。 The method for calculating p / q will be described in more detail. For the calculation, a sequence obtained by removing the sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m to the most C-terminal side from the domain (A) n motif to the C terminus of the domain sequence. (Hereinafter referred to as “array A”). First, in all REPs included in the sequence A, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in the four consecutive amino acid residues by 4 (number of amino acid residues). The average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of hydrophobicity index is 2.6 or more”, it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
　例えば、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が２０カ所抽出された場合（重複はなし）、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、連続する４アミノ酸残基（重複はなし）が２０含まれることになり、ｐは２０×４＝８０である。また、例えば、２つの「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が１アミノ酸残基だけ重複して存在する場合、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、７アミノ酸残基含まれることになる（ｐ＝２×４－１＝７。「－１」は重複分の控除である。）。例えば、図４に示したドメイン配列の場合、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が重複せずに７つ存在するため、ｐは７×４＝２８となる。また、例えば、図４に示したドメイン配列の場合、ｑは４＋５０＋４＋４０＋４＋１０＋４＋２０＋４＋３０＝１７０である（Ｃ末端側の最後に存在する（Ａ）ｎモチーフは含めない）。次に、ｐをｑで除すことによって、ｐ／ｑ（％）を算出することができる。図４の場合２８／１７０＝１６．４７％となる。 For example, when 20 “four consecutive amino acid residues with an average value of hydrophobicity index of 2.6 or more” are extracted (no overlap), the average value of the hydrophobicity index of four consecutive amino acid residues is 2 The region of .6 or more contains 20 consecutive 4 amino acid residues (no overlap), and p is 20 × 4 = 80. In addition, for example, when two “four consecutive amino acid residues having an average value of hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of four consecutive amino acid residues In the region where the average value of is 2.6 or more, 7 amino acid residues are included (p = 2 × 4-1 = 7, where “−1” is a deduction of duplicates). For example, in the case of the domain sequence shown in FIG. 4, there are seven “4 consecutive amino acid residues with an average value of hydrophobicity index of 2.6 or more” without duplication, and therefore p is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (the (A) n motif present at the end on the C-terminal side is not included). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.
　第４の実施形態に係る改変フィブロインにおいて、ｐ／ｑは、６．２％以上であることが好ましく、７％以上であることがより好ましく、１０％以上であることが更に好ましく、２０％以上であることが更により好ましく、３０％以上であることが更によりまた好ましい。ｐ／ｑの上限は、特に制限されないが、例えば、４５％以下であってもよい。 In the modified fibroin according to the fourth embodiment, p / q is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, and 20% or more. It is still more preferable that it is 30% or more. The upper limit of p / q is not particularly limited, but may be 45% or less, for example.
　第４の実施形態に係る改変フィブロインは、例えば、クローニングした天然由来のフィブロインのアミノ酸配列を、上記のｐ／ｑの条件を満たすように、ＲＥＰ中の１又は複数の親水性アミノ酸残基（例えば、疎水性指標がマイナスであるアミノ酸残基）を疎水性アミノ酸残基（例えば、疎水性指標がプラスであるアミノ酸残基）に置換すること、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入することにより、局所的に疎水性指標の大きい領域を含むアミノ酸配列に改変することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から上記のｐ／ｑの条件を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当する改変を行ってもよい。 The modified fibroin according to the fourth embodiment includes, for example, one or a plurality of hydrophilic amino acid residues in REP (for example, an amino acid sequence of a cloned naturally-derived fibroin so as to satisfy the above p / q condition) Substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or one or more hydrophobic amino acids during REP By inserting a residue, it can be obtained by locally modifying the amino acid sequence including a region having a large hydrophobicity index. Alternatively, for example, an amino acid sequence satisfying the above p / q conditions can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis. In any case, compared to naturally occurring fibroin, one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index and / or one or more amino acid residues in REP. In addition to modifications corresponding to insertion of amino acid residues having a large hydrophobicity index, modifications corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. .
　疎水性指標の大きいアミノ酸残基としては、特に制限はないが、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）が好ましく、バリン（Ｖ）、ロイシン（Ｌ）及びイソロイシン（Ｉ）がより好ましい。 The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A ) Are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
　第４の実施形態に係る改変フィブロインの別の具体的な例として、（４－ｉ）配列番号１５、配列番号１７若しくは配列番号１８で示されるアミノ酸配列、又は（４－ｉｉ）配列番号１５、配列番号１７若しくは配列番号１８で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As another specific example of the modified fibroin according to the fourth embodiment, (4-i) the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, or (4-ii) SEQ ID NO: 15, Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.
　（４－ｉ）の改変フィブロインについて説明する。配列番号１４で示されるアミノ酸配列は、天然由来のフィブロインの（Ａ）ｎモチーフ中のアラニン残基が連続するアミノ酸配列をアラニン残基が連続する数を５つになるよう欠失したものである。配列番号１５で示されるアミノ酸配列は、配列番号１４で示されるアミノ酸配列に対し、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を２カ所挿入し、かつ配列番号１４で示されるアミノ酸配列の分子量とほぼ同じとなるようにＣ末端側の一部のアミノ酸を欠失させたものである。配列番号１６で示されるアミノ酸配列は、配列番号１４で示されるアミノ酸配列に対し、各（Ａ）ｎモチーフのＣ末端側に２つのアラニン残基を挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、かつ配列番号１４で示されるアミノ酸配列の分子量とほぼ同じとなるようにＣ末端側の一部のアミノ酸を欠失させたものである。配列番号１７で示されるアミノ酸配列は、配列番号１６で示されるアミノ酸配列に対し、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を１カ所挿入したものである。配列番号１８で示されるアミノ酸配列は、配列番号１６で示されるアミノ酸配列に対し、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を２カ所挿入したものである。 The modified fibroin (4-i) will be described. The amino acid sequence shown in SEQ ID NO: 14 is an amino acid sequence in which alanine residues in the (A) n motif of (A) naturally derived fibroin are deleted so that the number of consecutive alanine residues is five. . The amino acid sequence shown in SEQ ID NO: 15 is inserted into the amino acid sequence shown in SEQ ID NO: 14 by two amino acid sequences (VLI) each consisting of 3 amino acid residues for every other REP, and shown in SEQ ID NO: 14. A part of amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence is almost the same. The amino acid sequence shown in SEQ ID NO: 16 is obtained by inserting two alanine residues on the C-terminal side of each (A) n motif with respect to the amino acid sequence shown in SEQ ID NO: 14, and further, some glutamine (Q) residues. A group is substituted with a serine (S) residue, and a part of amino acids on the C-terminal side is deleted so as to be approximately the same as the molecular weight of the amino acid sequence represented by SEQ ID NO: 14. The amino acid sequence represented by SEQ ID NO: 17 is obtained by inserting one amino acid sequence (VLI) consisting of 3 amino acid residues every other REP to the amino acid sequence represented by SEQ ID NO: 16. The amino acid sequence shown in SEQ ID NO: 18 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues into the amino acid sequence shown in SEQ ID NO: 16 every other REP.
　（４－ｉ）の改変フィブロインは、配列番号１５、配列番号１７又は配列番号１８で示されるアミノ酸配列からなるものであってもよい。 (4-i) The modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18.
　（４－ｉｉ）の改変フィブロインは、配列番号１５、配列番号１７又は配列番号１８で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（４－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin (4-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18. The modified fibroin of (4-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（４－ｉｉ）の改変フィブロインは、配列番号１５、配列番号１７又は配列番号１８で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつ最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であることが好ましい。 The modified fibroin of (4-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, and is located at the most C-terminal side (A) n In all REPs included in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence, the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues, and (A) When the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is q , P / q is preferably 6.2% or more.
　上述の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。 The above-mentioned modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal.
　タグ配列を含む改変フィブロインのより具体的な例として、（４－ｉｉｉ）配列番号１９、配列番号２０若しくは配列番号２１で示されるアミノ酸配列、又は（４－ｉｖ）配列番号１９、配列番号２０若しくは配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of modified fibroin containing a tag sequence, (4-iii) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (4-iv) SEQ ID NO: 19, SEQ ID NO: 20 or A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21 can be mentioned.
　配列番号１９、配列番号２０及び配列番号２１で示されるアミノ酸配列は、それぞれ配列番号１５、配列番号１７及び配列番号１８で示されるアミノ酸配列のＮ末端に配列番号５で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。 The amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 are the amino acid sequences represented by SEQ ID NO: 5 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18, respectively (His tag). Including a sequence and a hinge sequence).
　（４－ｉｉｉ）の改変フィブロインは、配列番号１９、配列番号２０若しくは配列番号２１で示されるアミノ酸配列からなるものであってもよい。 (4-iii) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
　（４－ｉｖ）の改変フィブロインは、配列番号１９、配列番号２０若しくは配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（４－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin (4-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (4-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（４－ｉｖ）の改変フィブロインは、配列番号１９、配列番号２０若しくは配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつ最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であることが好ましい。 The modified fibroin of (4-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) n In all REPs included in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence, the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues, and (A) When the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is q , P / q is preferably 6.2% or more.
　上述の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 The aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of host.
＜改変フィブロインの製造方法＞
　本実施形態に係る改変フィブロインは、例えば、当該改変フィブロインをコードする核酸配列と、当該核酸配列に作動可能に連結された１又は複数の調節配列とを有する発現ベクターで形質転換された宿主により、当該核酸を発現させることにより生産することができる。 <Method for producing modified fibroin>
The modified fibroin according to the present embodiment includes, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing the nucleic acid.
　改変フィブロインをコードする核酸の製造方法は、特に制限されない。例えば、天然のフィブロインをコードする遺伝子を利用して、ポリメラーゼ連鎖反応（ＰＣＲ）などで増幅しクローニングし、遺伝子工学的手法により改変する方法、又は、化学的に合成する方法によって、当該核酸を製造することができる。核酸の化学的な合成方法も特に制限されず、例えば、ＮＣＢＩのウェブデータベースなどより入手したフィブロインのアミノ酸配列情報をもとに、ＡＫＴＡ　ｏｌｉｇｏｐｉｌｏｔ　ｐｌｕｓ　１０／１００（ＧＥヘルスケア・ジャパン株式会社）などで自動合成したオリゴヌクレオチドをＰＣＲなどで連結する方法によって遺伝子を化学的に合成することができる。この際に、改変フィブロインの精製及び／又は確認を容易にするため、上記のアミノ酸配列のＮ末端に開始コドン及びＨｉｓ１０タグからなるアミノ酸配列を付加したアミノ酸配列からなる改変フィブロインをコードする核酸を合成してもよい。 The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, using the gene encoding natural fibroin, the nucleic acid is produced by a method such as amplification by polymerase chain reaction (PCR), cloning, modification by genetic engineering techniques, or chemical synthesis. can do. The method for chemically synthesizing nucleic acids is not particularly limited. For example, based on the amino acid sequence information of fibroin obtained from the NCBI web database, etc., AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) A gene can be chemically synthesized by a method of linking oligonucleotides that are synthesized automatically by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N terminus of the above amino acid sequence is synthesized. May be.
　調節配列は、宿主における改変フィブロインの発現を制御する配列（例えば、プロモーター、エンハンサー、リボソーム結合配列、転写終結配列等）であり、宿主の種類に応じて適宜選択することができる。プロモーターとして、宿主細胞中で機能し、改変フィブロインを発現誘導可能な誘導性プロモーターを用いてもよい。誘導性プロモーターは、誘導物質（発現誘導剤）の存在、リプレッサー分子の非存在、又は温度、浸透圧若しくはｐＨ値の上昇若しくは低下等の物理的要因により、転写を制御できるプロモーターである。 Regulatory sequences are sequences that control the expression of modified fibroin in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce expression of the modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value.
　発現ベクターの種類は、プラスミドベクター、ウイルスベクター、コスミドベクター、フォスミドベクター、人工染色体ベクター等、宿主の種類に応じて適宜選択することができる。発現ベクターとしては、宿主細胞において自立複製が可能、又は宿主の染色体中への組込みが可能で、改変フィブロインをコードする核酸を転写できる位置にプロモーターを含有しているものが好適に用いられる。 The type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like. As the expression vector, a vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.
　宿主として、原核生物、並びに酵母、糸状真菌、昆虫細胞、動物細胞及び植物細胞等の真核生物のいずれも好適に用いることができる。 As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
　原核生物の宿主の好ましい例として、エシェリヒア属、ブレビバチルス属、セラチア属、バチルス属、ミクロバクテリウム属、ブレビバクテリウム属、コリネバクテリウム属及びシュードモナス属等に属する細菌を挙げることができる。エシェリヒア属に属する微生物として、例えば、エシェリヒア・コリ等を挙げることができる。ブレビバチルス属に属する微生物として、例えば、ブレビバチルス・アグリ等を挙げることができる。セラチア属に属する微生物として、例えば、セラチア・リクエファシエンス等を挙げることができる。バチルス属に属する微生物として、例えば、バチルス・サチラス等を挙げることができる。ミクロバクテリウム属に属する微生物として、例えば、ミクロバクテリウム・アンモニアフィラム等を挙げることができる。ブレビバクテリウム属に属する微生物として、例えば、ブレビバクテリウム・ディバリカタム等を挙げることができる。コリネバクテリウム属に属する微生物として、例えば、コリネバクテリウム・アンモニアゲネス等を挙げることができる。シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属に属する微生物として、例えば、シュードモナス・プチダ等を挙げることができる。 Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
　原核生物を宿主とする場合、改変フィブロインをコードする核酸を導入するベクターとしては、例えば、ｐＢＴｒｐ２（ベーリンガーマンハイム社製）、ｐＧＥＸ（Ｐｈａｒｍａｃｉａ社製）、ｐＵＣ１８、ｐＢｌｕｅｓｃｒｉｐｔＩＩ、ｐＳｕｐｅｘ、ｐＥＴ２２ｂ、ｐＣｏｌｄ、ｐＵＢ１１０、ｐＮＣＯ２（特開２００２－２３８５６９号公報）等を挙げることができる。 When a prokaryotic host is used as a vector for introducing a nucleic acid encoding a modified fibroin, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like can be mentioned.
　真核生物の宿主としては、例えば、酵母及び糸状真菌（カビ等）を挙げることができる。酵母としては、例えば、サッカロマイセス属、ピキア属、シゾサッカロマイセス属等に属する酵母を挙げることができる。糸状真菌としては、例えば、アスペルギルス属、ペニシリウム属、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属等に属する糸状真菌を挙げることができる。 Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma and the like.
　真核生物を宿主とする場合、改変フィブロインをコードする核酸を導入するベクターとしては、例えば、ＹＥＰ１３（ＡＴＣＣ３７１１５）、ＹＥｐ２４（ＡＴＣＣ３７０５１）等を挙げることができる。上記宿主細胞への発現ベクターの導入方法としては、上記宿主細胞へＤＮＡを導入する方法であればいずれも用いることができる。例えば、カルシウムイオンを用いる方法〔Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ，６９，２１１０（１９７２）〕、エレクトロポレーション法、スフェロプラスト法、プロトプラスト法、酢酸リチウム法、コンピテント法等を挙げることができる。 When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC 37115) and YEp24 (ATCC 37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method, and the like.
　発現ベクターで形質転換された宿主による核酸の発現方法としては、直接発現のほか、モレキュラー・クローニング第２版に記載されている方法等に準じて、分泌生産、融合タンパク質発現等を行うことができる。 As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd edition, etc. .
　改変フィブロインは、例えば、発現ベクターで形質転換された宿主を培養培地中で培養し、培養培地中に当該改変フィブロインを生成蓄積させ、該培養培地から採取することにより製造することができる。宿主を培養培地中で培養する方法は、宿主の培養に通常用いられる方法に従って行うことができる。 The modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting from the culture medium. The method for culturing a host in a culture medium can be performed according to a method usually used for culturing a host.
　宿主が、大腸菌等の原核生物又は酵母等の真核生物である場合、培養培地として、宿主が資化し得る炭素源、窒素源及び無機塩類等を含有し、宿主の培養を効率的に行える培地であれば天然培地、合成培地のいずれを用いてもよい。 When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.
　炭素源としては、上記形質転換微生物が資化し得るものであればよく、例えば、グルコース、フラクトース、スクロース、及びこれらを含有する糖蜜、デンプン及びデンプン加水分解物等の炭水化物、酢酸及びプロピオン酸等の有機酸、並びにエタノール及びプロパノール等のアルコール類を用いることができる。窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム及びリン酸アンモニウム等の無機酸又は有機酸のアンモニウム塩、その他の含窒素化合物、並びにペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕及び大豆粕加水分解物、各種発酵菌体及びその消化物を用いることができる。無機塩類としては、例えば、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅及び炭酸カルシウムを用いることができる。 Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism. Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc. Organic acids and alcohols such as ethanol and propanol can be used. Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used. As inorganic salts, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
　大腸菌等の原核生物又は酵母等の真核生物の培養は、例えば、振盪培養又は深部通気攪拌培養等の好気的条件下で行うことができる。培養温度は、例えば、１５～４０℃である。培養時間は、通常１６時間～７日間である。培養中の培養培地のｐＨは３．０～９．０に保持することが好ましい。培養培地のｐＨの調整は、無機酸、有機酸、アルカリ溶液、尿素、炭酸カルシウム及びアンモニア等を用いて行うことができる。 Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture. The culture temperature is, for example, 15 to 40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
　また、培養中、必要に応じて、アンピシリン及びテトラサイクリン等の抗生物質を培養培地に添加してもよい。プロモーターとして誘導性のプロモーターを用いた発現ベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、ｌａｃプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはイソプロピル－β－Ｄ－チオガラクトピラノシド等を、ｔｒｐプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培地に添加してもよい。 Moreover, during the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter, and indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. An acid or the like may be added to the medium.
　発現させた改変フィブロインの単離、精製は通常用いられている方法で行うことができる。例えば、当該改変フィブロインが、細胞内に溶解状態で発現した場合には、培養終了後、宿主細胞を遠心分離により回収し、水系緩衝液に懸濁した後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー及びダイノミル等により宿主細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、改変フィブロインの単離精製に通常用いられている方法、すなわち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（ＤＥＡＥ）－セファロース、ＤＩＡＩＯＮ　ＨＰＡ－７５（三菱化成社製）等のレジンを用いた陰イオン交換クロマトグラフィー法、Ｓ－Ｓｅｐｈａｒｏｓｅ　ＦＦ（Ｐｈａｒｍａｃｉａ社製）等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の方法を単独又は組み合わせて使用し、精製標品を得ることができる。 Isolation and purification of the expressed modified fibroin can be performed by a commonly used method. For example, when the modified fibroin is expressed in a dissolved state in the cells, the host cells are recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a manton. A host cell is crushed with a Gaurin homogenizer, dynomill, etc., and a cell-free extract is obtained. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolation and purification of modified fibroin, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method by DE, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), or a resin such as S-Sepharose FF (manufactured by Pharmacia) Electrophoresis such as cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Use methods such as law alone or in combination It is possible to obtain a purified product.
　また、改変フィブロインが細胞内に不溶体を形成して発現した場合は、同様に宿主細胞を回収後、破砕し、遠心分離を行うことにより、沈殿画分として改変フィブロインの不溶体を回収する。回収した改変フィブロインの不溶体はタンパク質変性剤で可溶化することができる。該操作の後、上記と同様の単離精製法により改変フィブロインの精製標品を得ることができる。当該改変フィブロインが細胞外に分泌された場合には、培養上清から当該改変フィブロインを回収することができる。すなわち、培養物を遠心分離等の手法により処理することにより培養上清を取得し、その培養上清から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。 In addition, when the modified fibroin is expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to collect the modified fibroin insoluble substance as a precipitate fraction. The recovered insoluble form of modified fibroin can be solubilized with a protein denaturant. After the operation, a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture with a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
＜人造フィブロイン繊維の製造方法＞
　本実施形態に係る人造フィブロイン繊維は、公知の紡糸方法によって製造することができる。すなわち、例えば、改変フィブロインを主成分として含む人造フィブロイン繊維を製造する際には、まず、上述した方法に準じて製造した改変フィブロインをジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、又はヘキサフルオロイソプロパノール（ＨＦＩＰ）等の溶媒に、溶解促進剤としての無機塩と共に添加し、溶解してドープ液を作製する。次いで、このドープ液を用いて、湿式紡糸、乾式紡糸、乾湿式紡糸又は溶融紡糸等の公知の紡糸方法により紡糸して、目的とする人造フィブロイン繊維を得ることができる。好ましい紡糸方法としては、湿式紡糸又は乾湿式紡糸を挙げることができる。 <Manufacturing method of artificial fibroin fiber>
The artificial fibroin fiber according to the present embodiment can be produced by a known spinning method. That is, for example, when producing an artificial fibroin fiber containing a modified fibroin as a main component, first, the modified fibroin produced according to the above-described method is converted into dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), Alternatively, it is added to a solvent such as hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator and dissolved to prepare a dope solution. Next, the target artificial fibroin fiber can be obtained by spinning using this dope solution by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning. Preferred spinning methods include wet spinning or dry wet spinning.
　図５は、人造フィブロイン繊維を製造するための紡糸装置の一例を概略的に示す説明図である。図５に示す紡糸装置１０は、乾湿式紡糸用の紡糸装置の一例であり、押出し装置１と、未延伸糸製造装置２と、湿熱延伸装置３と、乾燥装置４とを有している。 FIG. 5 is an explanatory view schematically showing an example of a spinning device for producing artificial fibroin fibers. A spinning device 10 shown in FIG. 5 is an example of a spinning device for dry and wet spinning, and includes an extrusion device 1, an undrawn yarn production device 2, a wet heat drawing device 3, and a drying device 4.
　紡糸装置１０を使用した紡糸方法を説明する。まず、貯槽７に貯蔵されたドープ液６が、ギアポンプ８により口金９から押し出される。ラボスケールにおいては、ドープ液をシリンダーに充填し、シリンジポンプを用いてノズルから押し出しても良い。次いで、押し出されたドープ液６は、エアギャップ１９を経て、凝固液槽２０の凝固液１１内に供給され、溶媒が除去されて、改変フィブロインが凝固し、繊維状凝固体が形成される。次いで、繊維状凝固体が、延伸浴槽２１内の温水１２中に供給されて、延伸される。延伸倍率は供給ニップローラ１３と引き取りニップローラ１４との速度比によって決まる。その後、延伸された繊維状凝固体が、乾燥装置４に供給され、糸道２２内で乾燥されて、人造フィブロイン繊維が、巻糸体５として得られる。１８ａ～１８ｇは糸ガイドである。 A spinning method using the spinning device 10 will be described. First, the dope solution 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8. In the lab scale, the dope solution may be filled into a cylinder and extruded from a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied into the coagulating liquid 11 in the coagulating liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulated body is formed. Next, the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14. Thereafter, the stretched fibrous solidified body is supplied to the drying device 4 and dried in the yarn path 22, and an artificial fibroin fiber is obtained as the wound thread body 5. Reference numerals 18a to 18g denote thread guides.
　凝固液１１としては、脱溶媒できる溶液であればよく、例えば、メタノール、エタノール及び２－プロパノール等の炭素数１～５の低級アルコール、並びにアセトン等を挙げることができる。凝固液１１は、適宜水を含んでいてもよい。凝固液１１の温度は、０～３０℃であることが好ましい。口金９として、直径０．１～０．６ｍｍのノズルを有するシリンジポンプを使用する場合、押し出し速度は１ホール当たり、０．２～６．０ｍｌ／時間が好ましく、１．４～４．０ｍｌ／時間であることがより好ましい。凝固したタンパク質が凝固液１１中を通過する距離（実質的には、糸ガイド１８ａから糸ガイド１８ｂまでの距離）は、脱溶媒が効率的に行える長さがあればよく、例えば、２００～５００ｍｍである。未延伸糸の引き取り速度は、例えば、１～２０ｍ／分であってよく、１～３ｍ／分であることが好ましい。凝固液１１中での滞留時間は、例えば、０．０１～３分であってよく、０．０５～０．１５分であることが好ましい。また、凝固液１１中で延伸（前延伸）をしてもよい。凝固液槽２０は多段設けてもよく、また延伸は必要に応じて、各段、又は特定の段で行ってもよい。 The coagulation liquid 11 may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone. The coagulation liquid 11 may appropriately contain water. The temperature of the coagulation liquid 11 is preferably 0 to 30 ° C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the base 9, the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and 1.4 to 4.0 ml / hour More preferably it is time. The distance through which the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) has only to be a length that allows efficient desolvation, for example, 200 to 500 mm. It is. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min. The residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11. The coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as necessary.
　なお、人造フィブロイン繊維を得る際に実施される延伸は、例えば、上記した凝固液槽２０内で行う前延伸、及び延伸浴槽２１内で行う湿熱延伸の他、乾熱延伸も採用される。 In addition, as the stretching performed when obtaining the artificial fibroin fiber, for example, dry stretching is also adopted in addition to the pre-stretching performed in the coagulating liquid tank 20 and the wet heat stretching performed in the stretching bath 21.
　湿熱延伸は、温水中、温水に有機溶剤等を加えた溶液中、スチーム加熱中で行うことができる。温度としては、例えば、５０～９０℃であってよく、７５～８５℃が好ましい。湿熱延伸では、未延伸糸（又は前延伸糸）を、例えば、１～１０倍延伸することができ、２～８倍延伸することが好ましい。 Wet and hot stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, and in steam heating. The temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C. In wet heat drawing, undrawn yarn (or predrawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.
　乾熱延伸は、電気管状炉、乾熱板等を使用して行うことができる。温度としては、例えば、１４０℃～２７０℃であってよく、１６０℃～２３０℃が好ましい。乾熱延伸では、未延伸糸（又は前延伸糸）を、例えば、０．５～８倍延伸することができ、１～４倍延伸することが好ましい。 Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like. The temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C. In dry heat drawing, an undrawn yarn (or predrawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
　湿熱延伸及び乾熱延伸はそれぞれ単独で行ってもよく、またこれらを多段で、又は組み合わせて行ってもよい。すなわち、一段目延伸を湿熱延伸で行い、二段目延伸を乾熱延伸で行う、又は一段目延伸を湿熱延伸行い、二段目延伸を湿熱延伸行い、更に三段目延伸を乾熱延伸で行う等、湿熱延伸及び乾熱延伸を適宜組み合わせて行うことができる。 Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching. For example, wet heat stretching and dry heat stretching can be appropriately combined.
　最終的な延伸倍率は、その下限値が、未延伸糸（又は前延伸糸）に対して、好ましくは、１倍超、２倍以上、３倍以上、４倍以上、５倍以上、６倍以上、７倍以上、８倍以上、９倍以上のうちのいずれかであり、上限値が、好ましくは４０倍以下、３０倍以下、２０倍以下、１５倍以下、１４倍以下、１３倍以下、１２倍以下、１１倍以下、１０倍以下である。 The final draw ratio of the lower limit of the undrawn yarn (or predrawn yarn) is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times. Above, 7 times or more, 8 times or more, 9 times or more, and upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less 12 times or less, 11 times or less, and 10 times or less.
＜人造フィブロイン繊維＞
　本実施形態に係る人造フィブロイン繊維は、上述した改変フィブロインを紡糸したものであり、上述した改変フィブロインを主成分として含む。 <Artificial fibroin fiber>
The artificial fibroin fiber according to this embodiment is obtained by spinning the above-described modified fibroin and contains the above-described modified fibroin as a main component.
　本実施形態に係る人造フィブロイン繊維は、下記式で定義される収縮率が７％を超えるものであることが好ましい。これにより、従来知られているタンパク質繊維よりも高い収縮率で収縮することができ、また従来知られている合成繊維よりも穏やかな条件下で高い収縮率で収縮することができる。
　収縮率＝｛１－（沸点未満の水に接触させることを含む収縮加工を施した人造フィブロイン繊維の長さ／収縮加工を施す前の人造フィブロイン繊維の長さ）｝×１００（％） The artificial fibroin fiber according to this embodiment preferably has a shrinkage rate defined by the following formula exceeding 7%. Thereby, it can shrink | contract with a higher shrinkage rate than the conventionally known protein fiber, and can shrink | contract with a higher shrinkage rate under milder conditions than the conventionally known synthetic fiber.
Shrinkage rate = {1− (length of artificial fibroin fiber subjected to shrinkage processing including contact with water having less than boiling point / length of artificial fibroin fiber before being subjected to shrinkage processing)} × 100 (%)
　本実施形態に係る人造フィブロイン繊維の収縮率は、１５％以上であることが好ましく、２５％超であることがより好ましく、３２％以上であることが更に好ましく、４０％以上であることが更により好ましく、４８％以上であることが更によりまた好ましく、５６％以上であることが特に好ましく、６４％以上であることが特により好ましく、７２％以上であることが最も好ましい。収縮率の上限は、通常、８０％以下である。 The shrinkage rate of the artificial fibroin fiber according to this embodiment is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, and further preferably 40% or more. More preferably, it is 48% or more, still more preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more. The upper limit of shrinkage is usually 80% or less.
＜収縮加工＞
　収縮加工は、人造フィブロイン繊維を沸点未満の水と接触させること（以下、「接触ステップ」ともいう。）を含む。収縮加工は、水との接触後（接触ステップ後）の人造フィブロイン繊維を乾燥させること（以下、「乾燥ステップ」ともいう。）を更に含むものであってもよい。 <Shrink processing>
Shrinkage processing includes contacting artificial fibroin fiber with water having a boiling point (hereinafter also referred to as “contacting step”). The shrinking process may further include drying the artificial fibroin fiber after contact with water (after the contact step) (hereinafter also referred to as “drying step”).
　接触ステップで人造フィブロイン繊維に接触させる水の温度は、沸点未満であればよい。これにより、取扱い性及び収縮加工の作業性等が向上する。また、収縮時間を充分に短縮するという観点からは、水の温度の下限値が、１０℃以上であることが好ましく、４０℃以上であることがより好ましく、７０℃以上であることが更に好ましい。水の温度の上限値は９０℃以下であることが好ましい。 The temperature of the water to be brought into contact with the artificial fibroin fiber in the contact step may be less than the boiling point. Thereby, the handleability, the workability of shrinkage processing, and the like are improved. Further, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit value of the water temperature is preferably 10 ° C or higher, more preferably 40 ° C or higher, and further preferably 70 ° C or higher. . The upper limit of the water temperature is preferably 90 ° C. or lower.
　接触ステップにおいて、水を人造フィブロイン繊維に接触させる方法は、特に限定されない。当該方法として、例えば、人造フィブロイン繊維を水中に浸漬する方法、人造フィブロイン繊維に対して水を常温で又は加温したスチーム等の状態で噴霧する方法、及び人造フィブロイン繊維を水蒸気が充満した高湿度環境下に暴露する方法等が挙げられる。これらの方法の中でも、接触ステップにおいては、収縮時間の短縮化が効果的に図れると共に、加工設備の簡素化等が実現できることから、人造フィブロイン繊維を水中に浸漬する方法が好ましい。 In the contacting step, the method for bringing water into contact with the artificial fibroin fiber is not particularly limited. As the method, for example, a method of immersing the artificial fibroin fiber in water, a method of spraying water on the artificial fibroin fiber at room temperature or in a heated steam state, and the high humidity where the artificial fibroin fiber is filled with water vapor Examples include exposure to the environment. Among these methods, the method of immersing the artificial fibroin fiber in water is preferable in the contact step because the shrinkage time can be effectively shortened and the processing equipment can be simplified.
　接触ステップにおいて、人造フィブロイン繊維を弛緩させた状態で水に接触させると、人造フィブロイン繊維が、単に収縮するだけでなく、波打つように縮れてしまうことがある。このような縮れの発生を防止するために、例えば、人造フィブロイン繊維を繊維軸方向に緊張させ（引っ張り）ながら沸点未満の水と接触させるなど、人造フィブロイン繊維を弛緩させない状態で接触ステップを実施してもよい。 In the contact step, when the artificial fibroin fiber is brought into contact with water in a relaxed state, the artificial fibroin fiber may not only contract but also shrink like a wave. In order to prevent the occurrence of such shrinkage, for example, the contact step is performed in a state where the artificial fibroin fiber is not relaxed, for example, the artificial fibroin fiber is brought into contact with water below the boiling point while being tensioned (pulled) in the fiber axis direction. May be.
　乾燥ステップは、接触ステップを経た人造フィブロイン繊維を乾燥するステップである。乾燥は、例えば、自然乾燥でもよく、乾燥設備を使用して強制的に乾燥させてもよい。乾燥設備としては、接触型又は非接触型の公知の乾燥設備がいずれも使用可能である。また、乾燥温度も、例えば、人造フィブロイン繊維に含まれるタンパク質が分解したり、人造フィブロイン繊維が熱的損傷を受けたりする温度よりも低い温度であれば何ら限定されるものではないが、一般には、２０～１５０℃の範囲内の温度であり、５０～１００℃の範囲内の温度であることが好ましい。温度がこの範囲にあることにより、人造フィブロイン繊維の熱的損傷、又は人造フィブロイン繊維に含まれるタンパク質の分解が生ずることなく、人造フィブロイン繊維が、より迅速且つ効率的に乾燥される。乾燥時間は、乾燥温度等に応じて適宜に設定され、例えば、過乾燥による人造フィブロイン繊維の品質及び物性等への影響が可及的に排除され得る時間等が採用される。 The drying step is a step of drying the artificial fibroin fiber that has undergone the contact step. Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known drying equipment of contact type or non-contact type can be used. The drying temperature is not limited as long as it is lower than the temperature at which, for example, the protein contained in the artificial fibroin fiber is decomposed or the artificial fibroin fiber is thermally damaged. The temperature is in the range of 20 to 150 ° C., and the temperature is preferably in the range of 50 to 100 ° C. When the temperature is in this range, the artificial fibroin fiber is dried more quickly and efficiently without thermal damage of the artificial fibroin fiber or degradation of the protein contained in the artificial fibroin fiber. The drying time is appropriately set according to the drying temperature or the like. For example, a time during which the influence on the quality and physical properties of the artificial fibroin fiber due to overdrying can be eliminated as much as possible is adopted.
〔高収縮人造フィブロン繊維〕
　本発明に係る人造フィブロイン繊維は、高収縮が可能なため、例えば、上述した収縮加工（接触ステップ、及び必要に応じて乾燥ステップ）を経て、高収縮人造フィブロイン繊維を得ることができる。 [High shrinkage artificial fibron fiber]
Since the artificial fibroin fiber according to the present invention can be highly contracted, for example, the highly contracted artificial fibroin fiber can be obtained through the above-described shrinking process (contacting step and drying step as necessary).
　本実施形態に係る高収縮人造フィブロイン繊維は、上述のとおり定義される収縮率が７％を超えるものである。高収縮人造フィブロイン繊維の収縮率は、１５％以上であることが好ましく、２５％超であることがより好ましく、３２％以上であることが更に好ましく、４０％以上であることが更により好ましく、４８％以上であることが更によりまた好ましく、５６％以上であることが特に好ましく、６４％以上であることが特により好ましく、７２％以上であることが最も好ましい。収縮率の上限は、通常、８０％以下である。 The high-shrinkage artificial fibroin fiber according to this embodiment has a shrinkage rate defined as described above exceeding 7%. The shrinkage ratio of the high shrinkage artificial fibroin fiber is preferably 15% or more, more preferably more than 25%, further preferably 32% or more, and still more preferably 40% or more, More preferably, it is 48% or more, particularly preferably 56% or more, particularly preferably 64% or more, and most preferably 72% or more. The upper limit of shrinkage is usually 80% or less.
　図６は、人造フィブロイン繊維を収縮加工するための装置の一例を概略的に示す説明図である。図６に示す装置４０は、人造フィブロイン繊維を送り出すフィードローラ４２と、高収縮人造フィブロイン繊維３８を巻き取るワインダー４４と、接触ステップを実施するウォーターバス４６と、乾燥ステップを実施する乾燥機４８と、を有して構成されている。 FIG. 6 is an explanatory view schematically showing an example of an apparatus for shrinking an artificial fibroin fiber. The apparatus 40 shown in FIG. 6 includes a feed roller 42 that feeds out artificial fibroin fibers, a winder 44 that winds up the highly shrinkable artificial fibroin fibers 38, a water bath 46 that performs a contact step, and a dryer 48 that performs a drying step. , And is configured.
　より詳細には、フィードローラ４２は、人造フィブロイン繊維３６の巻回物が装着可能とされており、図示しない電動モータ等の回転によって、人造フィブロイン繊維３６の巻回物から人造フィブロイン繊維３６を連続的且つ自動的に送り出し得るようになっている。ワインダー４４は、フィードローラ４２から送り出された後、接触ステップと乾燥ステップを経て製造される高収縮人造フィブロイン繊維３８を、図示しない電動モータの回転によって連続的且つ自動的に巻き取り得るようになっている。なお、ここでは、フィードローラ４２による人造フィブロイン繊維３６の送出し速度と、ワインダー４４による高収縮人造フィブロイン繊維３８の巻取り速度とが、互いに独立して制御可能とされている。 More specifically, the feed roller 42 can be attached with a wound product of the artificial fibroin fiber 36, and the artificial fibroin fiber 36 is continuously formed from the wound product of the artificial fibroin fiber 36 by rotation of an electric motor (not shown). Can be delivered automatically and automatically. After being fed from the feed roller 42, the winder 44 can continuously and automatically wind up the high shrinkage artificial fibroin fiber 38 produced through the contact step and the drying step by the rotation of an electric motor (not shown). ing. Here, the feeding speed of the artificial fibroin fiber 36 by the feed roller 42 and the winding speed of the highly shrinkable artificial fibroin fiber 38 by the winder 44 can be controlled independently of each other.
　ウォーターバス４６と乾燥機４８は、フィードローラ４２とワインダー４４との間に、人造フィブロイン繊維３６の送り方向の上流側と下流側にそれぞれ並んで配置されている。なお、図６に示す装置４０は、フィードローラ４２からワインダー４４に向かって走行する人造フィブロイン繊維３６を中継するリレーローラ５０及び５２を有している。 The water bath 46 and the dryer 48 are arranged side by side between the feed roller 42 and the winder 44 on the upstream side and the downstream side in the feeding direction of the artificial fibroin fiber 36. The apparatus 40 shown in FIG. 6 has relay rollers 50 and 52 that relay the artificial fibroin fiber 36 that travels from the feed roller 42 toward the winder 44.
　ウォーターバス４６はヒータ５４を有し、このヒータ５４にて加温された湯４７が、ウォーターバス４６内に収容されている。また、ウォーターバス４６内には、テンションローラ５６が、湯４７中に浸漬された状態で設置されている。これにより、フィードローラ４２から送り出された人造フィブロイン繊維３６が、ウォーターバス４６内を、テンションローラ５６に巻き掛けられた状態で湯４７中に浸漬されつつ、ワインダー４４側に向かって走行するようになっている。なお、人造フィブロイン繊維３６の湯４７中への浸漬時間は、人造フィブロイン繊維３６の走行速度に応じて適宜にコントロールされる。 The water bath 46 has a heater 54, and hot water 47 heated by the heater 54 is accommodated in the water bath 46. A tension roller 56 is installed in the water bath 46 so as to be immersed in the hot water 47. As a result, the artificial fibroin fiber 36 fed out from the feed roller 42 travels toward the winder 44 while being immersed in the hot water 47 while being wound around the tension roller 56 in the water bath 46. It has become. The immersion time of the artificial fibroin fiber 36 in the hot water 47 is appropriately controlled according to the traveling speed of the artificial fibroin fiber 36.
　乾燥機４８は、一対のホットローラ５８を有している。一対のホットローラ５８は、ウォーターバス４６内から離脱してワインダー４４側に向かって走行する人造フィブロイン繊維３６が巻き掛け可能とされている。これにより、ウォーターバス４６内で湯４７に浸漬された人造フィブロイン繊維３６が、乾燥機４８内で一対のホットローラ５８にて加熱され、乾燥させられた後、ワインダー４４に向かって更に送り出されるようになっている。 The dryer 48 has a pair of hot rollers 58. The pair of hot rollers 58 can be wound around the artificial fibroin fiber 36 that moves away from the water bath 46 and travels toward the winder 44 side. As a result, the artificial fibroin fiber 36 immersed in the hot water 47 in the water bath 46 is heated by the pair of hot rollers 58 in the dryer 48 and dried, and then sent out further toward the winder 44. It has become.
　このような構造を有する装置４０を用いて、高収縮人造フィブロイン繊維３８を製造する際には、先ず、例えば、図５に示された紡糸装置１０を用いて紡糸された人造フィブロイン繊維３６の巻回物をフィードローラ４２に装着する。次に、フィードローラ４２から人造フィブロイン繊維３６を連続的に送り出して、ウォーターバス４６内で湯４７に浸漬させる。このとき、例えば、ワインダー４４の巻き取り速度をフィードローラ４２の送り出し速度よりも遅くしておく。これにより、人造フィブロイン繊維３６が、フィードローラ４２とワインダー４４との間で弛緩しないように緊張された状態で、湯４７との接触により収縮するため、縮れの発生を防止することができる。 When manufacturing the high-shrinkage artificial fibroin fiber 38 using the apparatus 40 having such a structure, first, for example, winding of the artificial fibroin fiber 36 spun using the spinning apparatus 10 shown in FIG. The circulation is mounted on the feed roller 42. Next, the artificial fibroin fiber 36 is continuously fed from the feed roller 42 and immersed in hot water 47 in the water bath 46. At this time, for example, the winding speed of the winder 44 is set slower than the feeding speed of the feed roller 42. Thereby, since the artificial fibroin fiber 36 is contracted by contact with the hot water 47 in a state where the artificial fibroin fiber 36 is tensioned so as not to relax between the feed roller 42 and the winder 44, occurrence of crimping can be prevented.
　次に、ウォーターバス４６内の湯４７中で収縮した人造フィブロイン繊維３６を、乾燥機４８の一対のホットローラ５８により加熱する。これにより、収縮した人造フィブロイン繊維３６を乾燥させて、高収縮人造フィブロイン繊維３８とする。このとき、フィードローラ４２の送出し速度とワインダー４４の巻取り速度との比率をコントロールすることで、人造フィブロイン繊維３６を更に収縮させることもできるし、長さを変化させないこともできる。そして、得られた高収縮人造フィブロイン繊維３８をワインダー４４にて巻き取って、高収縮人造フィブロイン繊維３８の巻回物を得る。 Next, the artificial fibroin fiber 36 contracted in the hot water 47 in the water bath 46 is heated by a pair of hot rollers 58 of the dryer 48. As a result, the contracted artificial fibroin fiber 36 is dried to obtain a highly contracted artificial fibroin fiber 38. At this time, by controlling the ratio between the feeding speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 36 can be further contracted, and the length can be kept unchanged. The obtained high-shrinkage artificial fibroin fiber 38 is wound up by a winder 44 to obtain a wound product of the high-shrinkage artificial fibroin fiber 38.
　なお、一対のホットローラ５８に代えて、図７に示されるような乾熱板６４等、単なる熱源のみからなる乾燥設備を用いて人造フィブロイン繊維３６を乾燥させてもよい。この場合にも、フィードローラ４２の送出し速度とワインダー４４の巻取り速度との互いの相対速度を、乾燥設備として一対のホットローラ５８を使用する場合と同様に調節することにより、人造フィブロイン繊維３６を更に収縮させることもできるし、長さを変化させないこともできる。ここでは、乾燥手段が乾熱板６４にて構成されることとなる。 In addition, it may replace with a pair of hot roller 58, and may dry the artificial fibroin fiber 36 using the drying equipment which consists only of mere heat sources, such as the dry heat board 64 as shown in FIG. Also in this case, by adjusting the relative speed of the feed speed of the feed roller 42 and the winding speed of the winder 44 in the same manner as in the case of using the pair of hot rollers 58 as the drying equipment, the artificial fibroin fiber 36 can be further shrunk or the length can be unchanged. Here, the drying means is constituted by the dry heat plate 64.
　上述のように、装置４０を用いることによって、目的とする高収縮人造フィブロイン繊維３８を自動的且つ連続的に、しかも極めて容易に製造することができる。 As described above, by using the apparatus 40, the intended high-shrinkage artificial fibroin fiber 38 can be manufactured automatically and continuously and extremely easily.
　図７は、人造フィブロイン繊維を収縮加工するための装置の別の例を概略的に示す説明図である。図７（ａ）は、当該装置に備わる、接触ステップを実施する加工装置を示し、図７（ｂ）は、当該装置に備わる、乾燥ステップを実施する乾燥装置を示す。図７に示される装置は、人造フィブロイン繊維３６に対する接触ステップを実施する加工装置６０と、加工装置６０にて接触ステップが実施された人造フィブロイン繊維３６を乾燥させる乾燥装置６２とを有し、それらが互いに独立した構造とされている。 FIG. 7 is an explanatory view schematically showing another example of an apparatus for shrinking artificial fibroin fibers. FIG. 7A shows a processing apparatus that performs the contact step provided in the apparatus, and FIG. 7B shows a drying apparatus that performs the drying step provided in the apparatus. The apparatus shown in FIG. 7 includes a processing device 60 that performs a contact step with respect to the artificial fibroin fiber 36, and a drying device 62 that dries the artificial fibroin fiber 36 subjected to the contact step with the processing device 60. Are independent of each other.
　より具体的には、図７（ａ）に示す加工装置６０は、図６に示された装置４０から乾燥機４８を省略して、フィードローラ４２とウォーターバス４６とワインダー４４とを、人造フィブロイン繊維３６の走行方向の上流から下流側に向かって順に並べて配置してなる構造を有している。このような加工装置６０は、フィードローラ４２から送り出された人造フィブロイン繊維３６を、ワインダー４４にて巻き取る前に、ウォーターバス４６内の湯４７中に浸漬させて、収縮させるようになっている。そして、湯４７中で収縮した人造フィブロイン繊維３６をワインダー４４にて巻き取るように構成されている。 More specifically, the processing device 60 shown in FIG. 7A omits the dryer 48 from the device 40 shown in FIG. 6, and replaces the feed roller 42, the water bath 46, and the winder 44 with the artificial fibroin. The fiber 36 has a structure in which the fibers 36 are arranged in order from the upstream side to the downstream side in the traveling direction. In such a processing device 60, the artificial fibroin fiber 36 fed from the feed roller 42 is immersed in hot water 47 in the water bath 46 and contracted before being wound up by the winder 44. . Then, the artificial fibroin fiber 36 contracted in the hot water 47 is configured to be wound up by the winder 44.
　図７（ｂ）に示す乾燥装置６２は、フィードローラ４２及びワインダー４４と、乾熱板６４とを有している。乾熱板６４は、フィードローラ４２とワインダー４４との間に、乾熱面６６が、人造フィブロイン繊維３６に接触し、且つその走行方向に沿って伸びるように配置されている。この乾燥装置６２では、前述したように、例えば、フィードローラ４２の送出し速度とワインダー４４の巻取り速度との比率をコントロールすることで、人造フィブロイン繊維３６を更に収縮させることもできるし、長さを変化させないこともできる。 7B includes a feed roller 42 and a winder 44, and a dry heat plate 64. The drying device 62 shown in FIG. The dry heat plate 64 is disposed between the feed roller 42 and the winder 44 so that the dry heat surface 66 contacts the artificial fibroin fiber 36 and extends along the traveling direction. In the drying device 62, as described above, the artificial fibroin fiber 36 can be further shrunk by controlling the ratio of the feed speed of the feed roller 42 and the winding speed of the winder 44, for example. It is also possible not to change the length.
　このような構造を有する装置を用いる場合には、例えば、先ず、人造フィブロイン繊維３６を、加工装置６０により収縮させた後、乾燥装置６２にて人造フィブロイン繊維３６を乾燥させることにより、目的とする高収縮人造フィブロイン繊維３８を製造することができる。 In the case of using an apparatus having such a structure, for example, first, the artificial fibroin fiber 36 is shrunk by the processing device 60, and then the artificial fibroin fiber 36 is dried by the drying device 62. High shrinkage man-made fibroin fiber 38 can be produced.
　なお、図７（ａ）に示された加工装置６０からフィードローラ４２とワインダー４４とを省略して、ウォーターバス４６のみで加工装置を構成してもよい。このような加工装置を有する装置を用いる場合には、例えば、高収縮人造フィブロイン繊維が、いわゆるバッチ式で製造されることとなる。 Note that the processing apparatus 60 may be configured with only the water bath 46 by omitting the feed roller 42 and the winder 44 from the processing apparatus 60 shown in FIG. In the case of using an apparatus having such a processing apparatus, for example, high-shrinkage artificial fibroin fibers are manufactured in a so-called batch system.
　以下、実施例等に基づいて本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples and the like. However, the present invention is not limited to the following examples.
〔（１）改変フィブロイン（人造フィブロイン）の製造〕
（改変フィブロインをコードする核酸の合成、及び発現ベクターの構築）
　配列番号７で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ３９９）、配列番号８で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ３８０）、配列番号９で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ４１０）、配列番号１３で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ７９９）を設計した。 [(1) Production of modified fibroin (artificial fibroin)]
(Synthesis of nucleic acid encoding modified fibroin and construction of expression vector)
Modified fibroin having the amino acid sequence represented by SEQ ID NO: 7 (PRT399), modified fibroin having the amino acid sequence represented by SEQ ID NO: 8 (PRT380), modified fibroin having the amino acid sequence represented by SEQ ID NO: 9 (PRT410), SEQ ID NO: A modified fibroin (PRT799) having the amino acid sequence shown by 13 was designed.
　配列番号７で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１で示されるアミノ酸配列から、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフを欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したアミノ酸配列に対し、Ｎ末端に配列番号５で示されるアミノ酸配列（Ｈｉｓタグを含む）を付加したものである。 The amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin, wherein (A) n motif is deleted every two from the N-terminal side to the C-terminal side. Further, an amino acid sequence represented by SEQ ID NO: 5 (including His tag) is added to the N-terminal to the amino acid sequence in which one [(A) n motif-REP] is inserted before the C-terminal sequence. .
　配列番号８で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１で示されるアミノ酸配列のＲＥＰ中の全てのＧＧＸをＧＱＸに置換したアミノ酸配列に対し、Ｎ末端に配列番号５で示されるアミノ酸配列（Ｈｉｓタグを含む）を付加したものである。 The amino acid sequence represented by SEQ ID NO: 8 is represented by SEQ ID NO: 5 at the N-terminal with respect to the amino acid sequence in which all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin are replaced with GQX. Added amino acid sequence (including His tag).
　配列番号９で示されるアミノ酸配列は、配列番号２で示されるアミノ酸配列のＲＥＰ中の全てのＧＧＸをＧＱＸに置換したアミノ酸配列に対し、Ｎ末端に配列番号５で示されるアミノ酸配列（Ｈｉｓタグを含む）を付加したものである。 The amino acid sequence represented by SEQ ID NO: 9 is the amino acid sequence represented by SEQ ID NO: 5 at the N-terminus (with His tag) compared to the amino acid sequence in which all GGXs in REP of the amino acid sequence represented by SEQ ID NO: 2 are replaced with GQX Included).
　配列番号１３で示されるアミノ酸配列は、配列番号９で示されるアミノ酸配列中に存在する２０個のドメイン配列の領域（但し、当該領域のＣ末端側の数アミノ酸残基が置換されている。）を４回繰り返した配列のＣ末端にＨｉｓタグが付加されたアミノ酸配列に対し、Ｎ末端に配列番号５で示されるアミノ酸配列（Ｈｉｓタグを含む）を付加したものである。 The amino acid sequence shown in SEQ ID NO: 13 is a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). The amino acid sequence represented by SEQ ID NO: 5 (including the His tag) is added to the N-terminus of the amino acid sequence in which the His tag is added to the C-terminus of the sequence obtained by repeating 4 times.
　設計した４種類の改変フィブロインをコードする核酸をそれぞれ合成した。当該核酸には、５’末端にＮｄｅＩサイト、終止コドン下流にＥｃｏＲＩサイトを付加した。これら４種類の核酸をクローニングベクター（ｐＵＣ１１８）にクローニングした。その後、同核酸をＮｄｅＩ及びＥｃｏＲＩで制限酵素処理して切り出した後、タンパク質発現ベクターｐＥＴ－２２ｂ（＋）に組換えて発現ベクターを得た。 The nucleic acids encoding the four types of designed modified fibroin were synthesized respectively. The nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon. These four types of nucleic acids were cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
（改変フィブロインの発現）
　得られたｐＥＴ－２２ｂ（＋）発現ベクターで、大腸菌ＢＬＲ（ＤＥ３）を形質転換した。当該形質転換大腸菌を、アンピシリンを含む２ｍＬのＬＢ培地で１５時間培養した。当該培養液を、アンピシリンを含む１００ｍＬのシード培養用培地（表２）にＯＤ６００が０．００５となるように添加した。培養液温度を３０℃に保ち、ＯＤ６００が５になるまでフラスコ培養を行い（約１５時間）、シード培養液を得た。 (Expression of modified fibroin)
Escherichia coli BLR (DE3) was transformed with the obtained pET-22b (+) expression vector. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium (Table 2) containing ampicillin so that the OD 600 was 0.005. The culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　当該シード培養液を５００ｍｌの生産培地（下記表３）を添加したジャーファーメンターにＯＤ６００が０．０５となるように添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持した。 The seed culture solution was added to a jar fermenter to which 500 ml of production medium (Table 3 below) was added so that the OD 600 was 0.05. The culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. The dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　生産培地中のグルコースが完全に消費された直後に、フィード液（グルコース４５５ｇ／１Ｌ、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　１２０ｇ／１Ｌ）を１ｍＬ／分の速度で添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持しながら、２０時間培養を行った。その後、１Ｍのイソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を培養液に対して終濃度１ｍＭになるよう添加し、目的とする改変フィブロインを発現誘導させた。ＩＰＴＧ添加後２０時間経過した時点で、培養液を遠心分離し、菌体を回収した。ＩＰＴＧ添加前とＩＰＴＧ添加後の培養液から調製した菌体を用いてＳＤＳ－ＰＡＧＥを行い、ＩＰＴＧ添加に依存した目的とする改変フィブロインに相当するサイズのバンドの出現により、目的とする改変フィブロインの発現を確認した。 Immediately after the glucose in the production medium was completely consumed, a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. The culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target modified fibroin. At the time when 20 hours passed after the addition of IPTG, the culture solution was centrifuged, and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of a band of a size corresponding to the target modified fibroin depending on the addition of IPTG Expression was confirmed.
（改変フィブロインの精製）
　ＩＰＴＧを添加してから２時間後に回収した菌体を２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｂｕｆｆｅｒ（ｐＨ７．４）で洗浄した。洗浄後の菌体を約１ｍＭのＰＭＳＦを含む２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）に懸濁させ、高圧ホモジナイザー（ＧＥＡ　Ｎｉｒｏ　Ｓｏａｖｉ社）で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）で洗浄した。洗浄後の沈殿物を１００ｍｇ／ｍＬの濃度になるように８Ｍ　グアニジン緩衝液（８Ｍ　グアニジン塩酸塩、１０ｍＭ　リン酸二水素ナトリウム、２０ｍＭ　ＮａＣｌ、１ｍＭ　Ｔｒｉｓ－ＨＣｌ、ｐＨ７．０）で懸濁し、６０℃で３０分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ（三光純薬株式会社製のセルロースチューブ３６／３２）を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収した。回収した凝集タンパク質から凍結乾燥機で水分を除き、目的とする改変フィブロインの凍結乾燥粉末を得た。 (Purification of modified fibroin)
The cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 60 ° C. And stirred for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). White aggregated protein obtained after dialysis was collected by centrifugation. Water was removed from the recovered aggregated protein with a freeze dryer to obtain the desired modified fibroin freeze-dried powder.
〔（２）人造フィブロイン繊維の製造〕
（ドープ液の調製）
　４．０質量％になるようにＬｉＣｌを溶解させたジメチルスルホキシド（ＤＭＳＯ）を溶媒として用意し、そこに改変フィブロインの凍結乾燥粉末を、濃度１８質量％又は２４質量％となるよう添加し（表４参照）、シェーカーを使用して３時間溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液を得た。 [(2) Manufacture of artificial fibroin fiber]
(Preparation of dope solution)
Prepare dimethyl sulfoxide (DMSO) in which LiCl is dissolved to 4.0% by mass as a solvent, and add a lyophilized powder of modified fibroin to the concentration of 18% by mass or 24% by mass (Table). 4) and dissolved using a shaker for 3 hours. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution.
（紡糸）
　得られた改変フィブロイン溶液をドープ液（紡糸原液）とし、図５に示す紡糸装置１０に準じた紡糸装置を用いた乾湿式紡糸によって、紡糸及び延伸された人造フィブロイン繊維を製造した。用いた紡糸装置は、図５に示す紡糸装置１０において、未延伸糸製造装置２（第１浴）及び湿熱延伸装置３（第３浴）の間に、更に第２の未延伸糸製造装置（第２浴）を備えるものである。乾湿式紡糸の条件は以下のとおりである。
　押出しノズル直径：０．２ｍｍ
　第１浴～第３浴中の液体及び温度：表４参照
　総延伸倍率：表４参照
　乾燥温度：６０℃ (spinning)
Using the obtained modified fibroin solution as a dope solution (spinning stock solution), spun and drawn artificial fibroin fibers were produced by dry and wet spinning using a spinning device similar to the spinning device 10 shown in FIG. The spinning device used is a spinning device 10 shown in FIG. 5, and a second undrawn yarn producing device (a first bath) and a wet heat drawing device 3 (third bath) between the undrawn yarn producing device 2 (first bath) and the second undrawn yarn producing device (3rd bath) A second bath). The dry and wet spinning conditions are as follows.
Extrusion nozzle diameter: 0.2 mm
Liquid and temperature in the first to third baths: see Table 4 Total draw ratio: see Table 4 Drying temperature: 60 ° C.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
〔（３）人造フィブロイン繊維の評価〕
（収縮性評価）
　製造例１～１９で得た各人造フィブロイン繊維について、収縮率を評価した。すなわち、各人造フィブロイン繊維に対して、沸点未満の水に接触させることからなる収縮加工１を施した場合の収縮率（以下、「一次収縮率」ということがある。）、及び沸点未満の水に接触させた後、室温で乾燥させることからなる収縮加工２を施した場合の収縮率（以下、「二次収縮率」ということがある。）を評価した。 [(3) Evaluation of artificial fibroin fiber]
(Shrinkage evaluation)
The shrinkage rate of each artificial fibroin fiber obtained in Production Examples 1 to 19 was evaluated. That is, each man-made fibroin fiber is subjected to shrinkage processing 1 comprising contact with water having a temperature lower than the boiling point (hereinafter sometimes referred to as “primary shrinkage rate”), and water having a temperature lower than the boiling point. Then, the shrinkage rate (hereinafter, also referred to as “secondary shrinkage rate”) was evaluated when shrinkage processing 2 comprising drying at room temperature was performed.
＜一次収縮率＞
　製造例１～１９で得た人造フィブロイン繊維の巻回物から、それぞれ、長さ３０ｃｍの複数本の試験用の人造フィブロイン繊維を切り出した。それら複数本の人造フィブロイン繊維を束ねて、繊度１５０デニールの人造フィブロイン繊維束を得た。各人造フィブロイン繊維束に０．８ｇの鉛錘を取り付け、その状態で各人造フィブロイン繊維束を表５～８に示す温度の水に１０分間浸漬した（収縮加工１）。その後、水中で各人造フィブロイン繊維束の長さを測定した。水中での人造フィブロイン繊維束の長さ測定は、人造フィブロイン繊維束の縮れを無くすために、人造フィブロイン繊維束に０．８ｇの鉛錘を取り付けたまま実施した。次いで、各人造フィブロイン繊維の一次収縮率（％）を、下記式Ｉに従って算出した。式Ｉ中、Ｌ０は収縮加工を施す前の人造フィブロイン繊維束の長さ（ここでは３０ｃｍ）を示し、Ｌｗは収縮加工１を施した人造フィブロイン繊維束の長さを示す。
　式Ｉ：収縮率（一次収縮率）＝｛１－（Ｌｗ／Ｌ０）｝×１００（％） <Primary shrinkage>
A plurality of artificial fibroin fibers for testing each having a length of 30 cm were cut out from the wound product of artificial fibroin fibers obtained in Production Examples 1 to 19. The plurality of artificial fibroin fibers were bundled to obtain an artificial fibroin fiber bundle having a fineness of 150 denier. 0.8 g of lead weight was attached to each artificial fibroin fiber bundle, and in this state, each artificial fibroin fiber bundle was immersed in water having a temperature shown in Tables 5 to 8 for 10 minutes (shrinkage processing 1). Thereafter, the length of each artificial fibroin fiber bundle was measured in water. The measurement of the length of the artificial fibroin fiber bundle in water was carried out with a 0.8 g lead weight attached to the artificial fibroin fiber bundle in order to eliminate the shrinkage of the artificial fibroin fiber bundle. Next, the primary shrinkage (%) of each artificial fibroin fiber was calculated according to the following formula I. In Formula I, L0 represents the length of the artificial fibroin fiber bundle (30 cm in this case) before being subjected to shrinkage processing, and Lw represents the length of the artificial fibroin fiber bundle subjected to shrinkage processing 1.
Formula I: Shrinkage rate (primary shrinkage rate) = {1− (Lw / L0)} × 100 (%)
＜二次収縮率＞
　一次収縮率の評価のための水への浸漬の後、人造フィブロイン繊維束を水中から取り出した。取り出した人造フィブロイン繊維束を、０．８ｇの鉛錘を取り付けたまま、室温で２時間おいて乾燥させた（収縮加工２）。乾燥後、各人造フィブロイン繊維束の長さを測定した。次いで、各人造フィブロイン繊維の二次収縮率（％）を、下記式ＩＩに従って算出した。式ＩＩ中、Ｌ０は収縮加工を施す前の人造フィブロイン繊維束の長さ（ここでは３０ｃｍ）を示し、Ｌｗｄは収縮加工２を施した人造フィブロイン繊維束の長さを示す。
　式ＩＩ：収縮率（二次収縮率）＝｛１－（Ｌｗｄ／Ｌ０）｝×１００（％） <Secondary shrinkage>
After immersion in water for evaluation of primary shrinkage, the artificial fibroin fiber bundle was removed from the water. The taken-out artificial fibroin fiber bundle was dried for 2 hours at room temperature with a 0.8 g lead weight attached (shrinkage processing 2). After drying, the length of each artificial fibroin fiber bundle was measured. Next, the secondary shrinkage (%) of each artificial fibroin fiber was calculated according to the following formula II. In Formula II, L0 represents the length of the artificial fibroin fiber bundle (30 cm in this case) before being subjected to shrinkage processing, and Lwd represents the length of the artificial fibroin fiber bundle subjected to shrinkage processing 2.
Formula II: Shrinkage rate (secondary shrinkage rate) = {1− (Lwd / L0)} × 100 (%)
　結果を表５～８に示す。
Figure JPOXMLDOC01-appb-T000005
 The results are shown in Tables 5-8.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　以上の結果から、本発明に係る人造フィブロイン繊維は、充分に高い収縮率を有することが明確に認識される。 From the above results, it is clearly recognized that the artificial fibroin fiber according to the present invention has a sufficiently high shrinkage rate.
　１…押出し装置、２…未延伸糸製造装置、３…湿熱延伸装置、４…乾燥装置、６…ドープ液、１０…紡糸装置、２０…凝固液槽、２１…延伸浴槽、３６…人造フィブロイン繊維、３８…高収縮人造フィブロイン繊維、４０…装置、４２…フィードローラ、４４…ワインダー、４６…ウォーターバス、４８…乾燥機、５４…ヒータ、５６…テンションローラ、５８…ホットローラ、６０…加工装置、６２…乾燥装置、６４…乾熱板。 DESCRIPTION OF SYMBOLS 1 ... Extrusion apparatus, 2 ... Undrawn yarn manufacturing apparatus, 3 ... Wet heat drawing apparatus, 4 ... Drying apparatus, 6 ... Dope liquid, 10 ... Spinning apparatus, 20 ... Coagulating liquid tank, 21 ... Drawing bath, 36 ... Artificial fibroin fiber 38 ... High shrinkage artificial fibroin fiber, 40 ... Equipment, 42 ... Feed roller, 44 ... Winder, 46 ... Water bath, 48 ... Dryer, 54 ... Heater, 56 ... Tension roller, 58 ... Hot roller, 60 ... Processing equipment 62 ... Drying device, 64 ... Dry heat plate.

Claims (4)

  1. 　改変フィブロインを含み、かつ下記式で定義される収縮率が７％を超える、人造フィブロイン繊維。
    　収縮率＝｛１－（沸点未満の水に接触させることを含む収縮加工を施した人造フィブロイン繊維の長さ／前記収縮加工を施す前の人造フィブロイン繊維の長さ）｝×１００（％）     An artificial fibroin fiber containing a modified fibroin and having a shrinkage rate defined by the following formula exceeding 7%.
    Shrinkage rate = {1− (length of artificial fibroin fiber subjected to shrinking process including contact with water having less than boiling point / length of artificial fibroin fiber before performing the shrinking process)} × 100 (%)
  2. 　前記改変フィブロインが、改変クモ糸フィブロインである、請求項１に記載の人造フィブロイン繊維。 The artificial fibroin fiber according to claim 1, wherein the modified fibroin is a modified spider silk fibroin.
  3. 　前記水の温度が、１０～９０℃である、請求項１又は２に記載の人造フィブロイン繊維。 The artificial fibroin fiber according to claim 1 or 2, wherein the temperature of the water is 10 to 90 ° C.
  4. 　前記収縮加工が、前記水との接触後に乾燥させることを含む、請求項１～３のいずれか一項に記載の人造フィブロイン繊維。 The artificial fibroin fiber according to any one of claims 1 to 3, wherein the shrinking process includes drying after contact with the water.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3476859A4 (en) * 2016-06-23 2019-12-11 Spiber Inc. MODIFIED FIBROIN
WO2020067553A1 (en) * 2018-09-28 2020-04-02 株式会社島精機製作所 Protein spun yarn manufacturing method
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