highly contracted artificial fibroin fiber, and methods for producing and contracting an artificial

专利摘要:
the present invention relates to a contracted synthetic fibroin fiber comprising modified fibroin, in which the percentage of contraction defined by the following equation exceeds 7%. contraction percentage = {1- (length of synthetic fibroin fiber contracted / length of synthetic fibroin fiber after spinning and before contact with water)} × 100 (%).
公开号:BR112019018454A2
申请号:R112019018454
申请日:2018-03-05
公开日:2020-04-14
发明作者:Nakamura Hiroyuki；Sugahara Junichi；Morita Keisuke
申请人:Spiber Inc；
IPC主号:

专利说明:

HIGHLY CONTRACTED ARTIFICIAL FIBROIN FIBER, AND,
METHODS FOR PRODUCING AND CONTRACTING A FIBER OF
ARTIFICIAL FIBROIN
TECHNICAL FIELD [001] The present invention relates to a highly contracted artificial fibroin fiber, a method of producing it, and a method to contract an artificial fibroin fiber.
BACKGROUND OF THE INVENTION [002] Generally, fibers used for clothing, bedding and the like are required to have a high level of tactile properties. As a fiber that satisfies sufficient tactile properties and having high final properties, silk is known as a type of natural fibroin fiber.
[003] In addition, softness, heat retention properties and the like are also required for fibers used for clothing, bedding and the like. In this way, for example, silk used for clothing, bedding and the like can be silk that has undergone a contraction process to increase the volume, whereby flexibility and heat retention properties are conferred.
[004] However, synthetic fibers such as polyester fibers, polyamide fibers and acrylic fibers are generally used for clothing, bedding and the like, and these synthetic fibers have a contraction percentage of 40% or more when they are brought into contact with boiling water (Patent Literature 1).
CITATION LIST
Patent Literature [005] [Patent Literature 1] Non-Patent Specification Report
Examined Japanese No. 2009-121003
SUMMARY OF THE INVENTION
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PROBLEMS TO BE SOLVED BY THE INVENTION [006] However, silk has only a small percentage of contraction when placed in contact with water only.
[007] In addition, a method of contraction described in Patent Literature 1 involves a great risk due to the handling of boiling water at high temperature.
[008] An object of the present invention is to provide a highly contracted artificial fibroin fiber that has a sufficiently high contraction percentage, has excellent tactile properties and flexibility, and can be safely produced; and a method of producing it. Another objective of the present invention is to provide a method for contracting an artificial fibroin fiber, which is capable of safely obtaining a sufficiently high percentage of contraction.
MEANS TO SOLVE PROBLEMS [009] The present invention relates, for example, to each of the following inventions.
[1] [0010] An artificial fibroin fiber contracted including:
a modified fibroin, in which the percentage of contraction defined by the following equation exceeds 7%.
Contraction percentage = {1- (length of artificial fibroin fiber contracted / length of artificial fibroin fiber before being brought into contact with water after spinning)} x 100 (%) [2] [0011] The artificial fibroin fiber highly contracted according to [1], where the modified fibroin is a modified spider silk fibroin.
[3]
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3/59 [0012] The highly contracted artificial fibroin fiber according to [1] or [2], which is contracted when it is placed in contact with water below a boiling point.
[4] [0013] The highly contracted artificial fibroin fiber according to [3], where a water temperature is 10 ° C to 90 ° C.
[5] [0014] The highly contracted artificial fibroin fiber according to [3] or [4], which is additionally contracted by drying after being placed in contact with water.
[6] [0015] A method for producing a highly contracted artificial fibroin fiber, including:
a step of contracting an artificial fibroin fiber containing a modified fibroin by placing the artificial fibroin fiber in contact with water below a boiling point, where a percentage of contraction defined by the following equation exceeds 7%.
Contraction percentage = {1- (length of artificial fibroin fiber contracted / length of artificial fibroin fiber before being brought into contact with water after spinning)} x 100 (%) [7] [0016] The method for producing a highly contracted artificial fibroin fiber according to [6], wherein the modified fibroin is a modified spider silk fibroin.
[8] [0017] The method for producing a highly contracted artificial fibroin fiber according to [6] or [7], where a water temperature is 10 ° C to 90 ° C.
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4/59 [9] [0018] The method for producing a highly contracted artificial fibroin fiber according to any one of [6] to [8], wherein the step of contracting the artificial fibroin fiber further includes drying the fiber of artificial fibroin after being placed in contact with water.
[10] [0019] A method for contracting an artificial fibroin fiber, including:
a step of contracting an artificial fibroin fiber containing a modified fibroin by placing the artificial fibroin fiber in contact with water below a boiling point, where a percentage of contraction defined by the following equation exceeds 7%.
Contraction percentage = {1- (length of artificial fibroin fiber contracted / length of artificial fibroin fiber before being brought into contact with water after spinning)} x 100 (%) [11] [0020] The method for contracting a artificial fibroin fiber according to [10], wherein the modified fibroin is a modified spider silk fibroin.
[12] [0021] The method for contracting an artificial fibroin fiber according to [10] or [11], where a water temperature is 10 ° C to 90 ° C.
[13] [0022] The method of contracting an artificial fibroin fiber according to any one of [10] to [12], wherein the step of contracting the artificial fibroin fiber further includes drying the artificial fibroin fiber after being put in contact with water.
EFFECTS OF THE INVENTION
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According to the present invention, it is possible to provide a highly contracted artificial fibroin fiber that has a sufficiently high contraction percentage, has excellent tactile properties and flexibility, and can be safely produced; and a method of producing it. According to the present invention, it is also possible to provide a method for contracting an artificial fibroin fiber, which is capable of safely obtaining a sufficiently high percentage of contraction.
BRIEF DESCRIPTION OF THE DRAWINGS [0024] FIG. 1 is a schematic diagram showing a domain sequence for a modified fibroin.
[0025] FIG. 2 is a graph showing a distribution of z / w (%) values for naturally occurring fibroin.
[0026] FIG. 3 is a graph showing a distribution of x / y (%) values for naturally occurring fibroin.
[0027] FIG. 4 is an explanatory view showing schematically an example of a spinning apparatus for producing artificial fibroin fibers.
[0028] FIG. 5 is an explanatory view showing schematically an example of a production apparatus for producing highly contracted artificial fibroin fibers.
[0029] FIG. 6 is an explanatory view showing schematically an example of a production apparatus for producing highly contracted artificial fibroin fibers.
MODALITIES FOR CARRYING OUT THE INVENTION [0030] In the following, modalities for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
[Highly contracted artificial fibroin fiber] [0031] A highly contracted artificial fibroin fiber according
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6/59 with the present invention is a contracted artificial fibroin fiber including a modified fibroin. In the highly contracted artificial fibroin fiber according to the present modality, a percentage of contraction defined by the following equation exceeds 7%.
Contraction percentage = {1- (length of artificial fibroin fiber contracted / length of artificial fibroin fiber before being brought into water after spinning)} x 100 (%) <Modified fibroin>
[0032] Fibroin modified according to the present modality is a protein including a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The modified fibroin may additionally have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence, although not limited to this, are typically regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of amino acids of about 100 residues.
[0033] The term "modified fibroin" as used in this specification means a synthetically produced fibroin (an artificial fibroin). The modified fibroin can be a fibroin whose domain sequence is different from the naturally occurring amino acid sequence of fibroin, or it can be a fibroin whose amino acid sequence is the same as that of naturally occurring fibroin. The term "naturally occurring fibroin" as used here is also a protein including a domain sequence represented by Formula 1: [(A) _n motif-REP] _m .
[0034] The "modified fibroin" can be a fibroin in which the naturally occurring fibroin amino acid sequence is used as is; it can be a fibroin; a fibroin whose amino acid sequence has been modified based on the occurring fibroin amino acid sequence
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7/59 natural (for example, a fibroin whose amino acid sequence has been modified by altering a cloned naturally occurring fibroin genetic sequence); or an artificially designed and synthesized fibroin regardless of naturally occurring fibroin (for example, a fibroin having a desired amino acid sequence chemically synthesizing a nucleic acid that encodes the projected amino acid sequence), provided that it has the specified amino acid sequence herein invention.
[0035] The term "domain sequence" as used here refers to an amino acid sequence that produces a crystalline region (which typically corresponds to motif (A) _n of an amino acid sequence) and an amorphous region (which typically corresponds to REP of an amino acid sequence) peculiar to fibroin and means an amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m . Reason (A) _n indicates an amino acid sequence mainly including an alanine residue, where n is an integer from 2 to 20, is preferably 4 to 20, is more preferably 8 to 20, is even more preferably 10 to 20, it is also even more preferably 4 to 16, it is also even more preferably 8 to 16, and it is particularly preferably preferably 10 to 16. In addition, it is sufficient provided that a percentage of alanine residues is 40% or more with respect to the number total amino acid residues in motif (A) _n , but is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90%, and can be 100% (which means that motif (A) does _not consist only of alanine residues). REP represents a sequence of amino acids consisting of 2 to 200 amino acid residues. m represents an integer from 2 to 300. A plurality of motifs (A) _n can be the same sequence of amino acids or different sequences of amino acids. A plurality of REPs can be the same amino acid sequence or different amino acid sequences.
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Specific examples of proteins derived from the large nasogastric silkworm include a protein containing the amino acid sequence (PRT410) shown in SEQ ID NO: 9.
[0036] Fibroin modified in accordance with the present modality can be obtained, for example, by making modifications to an amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or a plurality of amino acid residues with respect to the genetic sequence of a naturally occurring cloned fibroin. Replacement, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, they can be performed according to a method described in the literature such as Nucleic Acid Res. 10, 6487 (1982), and Methods in Enzymology, 100, 448 (1983).
[0037] Naturally occurring fibroma is a protein including a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , specifically, for example, a fibroin produced by insects or spiders. [0038] Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pemyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea mylitta, and Antheraea mylitta assama; and Hornet silk proteins discharged by Vespa simillima xanthoptera larvae.
[0039] A more specific example of fibroin produced by insects may be an L silkworm fibroin chain (GenBank Access No. M76430 (base sequence), AAA27840.1 (amino acid sequence)).
[0040] Examples of fibroin produced by spiders include spider silk proteins produced by spiders belonging to the Araneus genus such as Araneus ventricosus, Araneus diadematus, Araneus pinguis, Araneus pentagrammicus and Araneus nojimai, spiders belonging to the
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9/59 Neoscona genus such as Neoscona scylla, Neoscona nautica, Neoscona advances and Neoscona scylloides, spiders that belong to the genus Promts such as Pronous minutes, spiders that belong to the genus Cyrtarachne such as Cyrtarachne bufo and Cyrtarachne inaequalis, spiders that belong to the genera Ga such as Gasteracantha kuhli and Gasteracantha mammosa, spiders that belong to the genus Ordgarius such as Ordgarius hobsoni and Ordgarius sexspinosus, spiders that belong to the genus Argiope such as Argiope amoena, Argiope minuta and Argiope bruennich, spiders that belong to the genus Arachnura log as spiders belonging to the genus Acusilas such as Acusilas coccineus, spiders belonging to the genus Cytophora such as Cyrtophora moluccensis, Cyrtophora exanthematica and Cyrtophora unicolor, spiders belonging to the genus Poltys such as Poltys illepidus, spiders belonging to the genus Cyclosa such as Cyclosa and Cyclosa Cyclosa sedeculata, Cyclosa va llata and Cyclosa atrata, and spiders belonging to the Chorizopes genus such as Chorizopes nipponicus; and spider silk proteins produced by spiders belonging to the genus Tetragnatha such as Tetragnatha praedonia, Tetragnatha maxillosa, Tetragnatha extensa and Tetragnatha squamata, spiders belonging to the Leucauge genus such as magnificent Leucauge, Leucauge blanda and Leucauge subblanda, spiders that belong to the genus subbland Nephila such as Nephila clavata and Nephila pilipes, spiders belonging to the genus Menosira such as Menosira ornata, spiders belonging to the genus Dyschiriognatha such as Dyschiriognatha tenera, spiders belonging to the genus Latrodectus such as Latrodectus mactans, Latrodectususselodus tresectus lattodecus lattee and lattee , and spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include traction fiber proteins such as MaSp (MaSpl and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSpl and MiSp2).
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10/59 [0041] More specific examples of fibroin produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank Accession Number 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 spidroin 1 [derived from Nephila clavipes] (Number GenBank Access Code AAC04504 (amino acid sequence), U37520 (base sequence)), main ampoule spidroin [derived from Latrodectus hesperus] (GenBank Access Number ABR68856 (amino acid sequence)), EF595246 (base sequence) ), dragline 2 of the dragline silk protein [derived from Nephila clavata] (GenBank Accession Number AAL32472 (amino acid sequence), AF441245 (base sequence)), spiroin 1 from the main ampoule [derived from Euprosthenops australis] (Number of GenBank access CAJ00428 (sequence of amino acids), AJ973155 (base sequence)) and spidroin 2 of the main ampoule [Euprosthenops australis] (GenBank Accession Number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), secondary protein silk protein [Nephila clavipes] (GenBank Accession Number AAC14589.1 (amino acid sequence)), Secondary ampoule silk protein 2 [Nephila clavipes] (GenBank Accession Number AAC14591.1 (amino acid sequence)), and type protein secondary ampoule spidroína [Nephilengys cruentata] (GenBank Accession Number ABR37278.1 (amino acid sequence)).
[0042] As a more specific example of naturally occurring fibroin, fibroin in which sequence information is recorded on the GenBank NCBI can be mentioned in addition. For example, it can be confirmed by extracting sequences in which spidroin, ampoule, fibroin, "silk and polypeptide", or "silk and protein" are described as a DEFINITION keyword among the sequences containing INV as DIVISION between
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11/59 sequence information registered to GenBank NCBI, sequences in which a specific character sequence is described by the CDS, or sequences in which a specific character sequence is described by the
SOURCE in FABRIC TYPE.
[0043] The fibroin modified in accordance with the present modality may be modified silk fibroin (where the amino acid sequence of the silk protein produced by silkworm is modified), or it may be a modified spider silk fibroin (in that the amino acid sequence of a spider silk protein produced by spiders is modified).
[0044] Specific example of the modified fibroin according to the present modality includes a modified fibroin derived from the large nasogasus dragline protein produced in a major spider strain (the modified fibroin according to the first modality), a modified fibroin having a reduced content glycine residue (a modified fibroin according to the second modality), a modified fibroin in which the motif content (A) _n is reduced (a modified fibroin according to the third modality), and a modified fibroin in which the glycine residue content and motif content (A) _n are reduced (a fibroin modified according to the fourth modality).
[0045] Examples of the modified fibroin according to the first modality include a protein including a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In fibroin modified according to the first modality, in Formula 1, n is preferably an integer from 3 to 20, more preferably an integer from 4 to 20, even more preferably an integer from 8 to 20, and also even more preferably an integer from 10 to 20, also even more preferably an integer from 4 to 16, in particular preferably an integer from 8 to 16, and above all preferably an integer from 10 to 16. In the modified fibroin of according to
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In the first embodiment, in Formula 1, the number of amino acid residues that make up the REP is preferably 10 to 200 residues, more preferably 10 to 150 residues, even more preferably 20 to 100 residues, and also even more preferably 20 to 75 residues. waste. In fibroin modified according to the first modality, a total number of residues of glycine residues, serine residues, and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more, with respect to the total number of amino acid residues.
[0046] The modified fibroin according to the first modality includes a unit of the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m , and can be a polypeptide which is an amino acid sequence having a homology of 90% or more with the amino acid sequence whose C-terminal sequence is shown in any of SEQ ID NOs: 12 to 14, or the amino acid sequence shown in any of SEQ ID NOs: 12 to 14.
[0047] The amino acid sequence shown in SEQ ID NO: 12 is identical to the amino acid sequence consisting of 50 amino acid residues at the C-terminus of the ADF3 amino acid sequence (GI: 1263287, NCBI); the amino acid sequence shown in SEQ ID NO: 13 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 12; and the amino acid sequence shown in SEQ ID NO: 14 is identical to the amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 12.
[0048] A more specific example of the modified fibroin according to the first modality may be a modified fibroin including (1-i), an amino acid sequence shown in SEQ ID NO: 15 or (1-ii), a
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13/59 amino acid sequence having 90% or more identity sequence with the amino acid sequence shown in SEQ ID NO: 15. The identity sequence is preferably 95% or more.
[0049] The amino acid sequence shown in SEQ ID NO: 15 is an amino acid sequence in which the first to the thirteenth repeating regions are increased to approximately double, and which is fined in such a way that the translation is completed in 1. 154- amino acid residue, in the amino acid sequence of ADF3 in which the amino acid sequence consisting of initiation codon, HislO tag, and HRV3C protease recognition site (Human rhinovirus 3C protease) (SEQ ID NO: 16 ) are added to the N-termination. The amino acid sequence at the C-terminus of the amino acid sequence shown in SEQ ID NO: 15 is identical to the amino acid sequence shown in SEQ ID NO: 3 [0050] The modified fibroin of (1-i) may consist of the amino acid sequence presented in SEQ ID NO: 15.
[0051] Fibroin modified according to the second modality has an amino acid sequence whose domain sequence has a reduced content of glycine residues compared to naturally occurring fibroin. It can be said that the modified fibroin may have at least one amino acid sequence corresponding to the replacement of one or a plurality of glycine residues in REP with another amino acid residue, compared to naturally occurring fibroin.
[0052] The modified fibroin according to the second modality can be a modified fibroin in which the domain sequence has at least one motif sequence selected from GGX and GPGXX (where X represents an amino acid residue other than glycine) in REP , at least one amino acid sequence corresponding to the replacement of a glycine residue in one or a plurality of motif sequences with a
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14/59 another amino acid residue, compared to naturally occurring fibroin.
[0053] The modified fibroin according to the second modality can be a modified fibroin in which the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue is
10% or more with respect to the total motif string.
[0054] The modified fibroin according to the second modality can be a modified fibroin that includes a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and has an amino acid sequence in which z / w is 30% or more, or has an amino acid sequence in which z / w is 50.9% or more, in the case where 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 of motif (A) _n located on the C-terminal side up to the C-terminus of the domain sequence from the domain sequence is defined as z, and the total number of amino acid residues in the sequence excluding motif sequence (A) _n located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence is defined as w. This is sufficient as long as the number of alanine residues is 83% or more with respect to the total number of amino acid residues in reason (A) _n , but is preferably 86% or more, more preferably 90% or more, also more preferably 95% or more, and even more preferably 100% (which means that motif (A) does _not consist only of alanine residues).
[0055] The modified fibroin according to the second embodiment is preferably a modified fibroin in which the ratio of the amino acid sequence content consisting of XGX is increased by replacing a glycine residue of the GGX motif with another amino acid residue. In fibroin modified according to the second modality, the ratio of the content of the amino acid sequence consisting of GGX in the domain sequence is
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15/59 preferably 30% or less, more preferably 20% or less, also more preferably 10% or less, still more preferably 6% or less, also additionally preferably 4% or less, and in particular preferably preferably 2% or less . The ratio of the content of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the method as the method of calculating the ratio of the content (z / w) of the amino acid sequence consisting of XGX described below.
[0056] The z / w calculation method will be described in more detail. First, from the domain sequence, an amino acid sequence consisting of XGX is extracted from all the REPs contained in the sequence excluding the sequence of motif (A) _n located on the most C-terminal side until the C-terminus of the domain sequence. The total number of amino acid residues that make up XGX is z. For example, in the case where 50 amino acid sequences consisting of XGX are extracted (there is no overlap), z is 50x3 = 150. Also, for example, in the case where X (central X) contained in two XGXs exists as in the case of the sequence of amino acids consisting of XGXGX, it is calculated by subtracting the overlap portion (in the case of XGXGX, it has 5 amino acid residues). w is the total number of amino acid residues contained in the sequence excluding the sequence of motif (A) _n located on the C-terminal side up to the C-terminus of the domain sequence from 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 motif (A) _n located on the more C-terminal side). Then z / w (%) can be calculated by dividing z by w.
[0057] Here, z / w in naturally occurring fibroin will be described.
First, as previously described, 663 types of fibroins (415 types of fibroins derived from spiders among them) were extracted confirming
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16/59 fibroins with amino acid sequence information registered in GenBank NCBI by an exemplified method, z / w was calculated using the aforementioned calculation method of naturally occurring fibroin amino acid sequences that include a domain sequence represented by Formula 1: [(A) _n motif-REP] _m and in which the ratio of the content of amino acid sequence consisting of GGX in the fibroin is 6% or less, among all extracted fibroins. The results are shown in FIG. 2. In FIG. 2, the horizontal geometric axis represents z / w (%) and the vertical geometric axis represents frequency. As is apparent from FIG. 2, z / w in naturally occurring fibroin is less than 50.9% (highest, 5.086%).
[0058] In the modified fibroin according to the second embodiment, z / w is preferably 50.9% or more, more preferably 56.1% or more, also more preferably 58.7% or more, even more preferably 70% or more, and also additionally preferably 80% or more. The upper limit of z / w is not particularly limited, but it can be 95% or less, for example.
[0059] Fibroin modified according to the second modality can be obtained, for example, by replacing and modifying at least a part of a base sequence that encodes a glycine residue of the cloned naturally occurring fibroin genetic sequence, in order to encode another amino acid residue. At this time, a glycine residue in the GGX motif and GPGXX motif can be selected as the glycine residue to be modified, and substitution can be carried out in such a way that z / w is 50.9% or more. Alternatively, the modified fibroin according to the modality can also be obtained, for example, by designing an amino acid sequence that satisfies each of the previous modalities of the naturally occurring fibroin amino acid sequence, and chemically synthesizing a nucleic acid that encodes the sequence of projected amino acids. In any case, in addition to the
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17/59 modification corresponding to the replacement of a glycine residue in REP with another amino acid residue of the naturally occurring fibroin amino acid sequence, modification of the amino acid sequence corresponding to the replacement, deletion, insertion and / or addition of one or one plurality of amino acid residues can be performed.
[0060] The other amino acid residue mentioned above is not particularly limited, as long as it is an amino acid residue other than a glycine residue, but it is preferably a hydrophobic amino acid residue such as a valine (V) residue, a leucine residue (L), isoleucine residue (I), methionine residue (M), proline residue (P), phenylalanine residue (F), or tryptophan residue (W), or hydrophilic amino acid residue such as a glutamine residue (Q), an asparagine residue (N), a serine residue (S), a lysine residue (K), or a glutamic acid residue (E), between the which most preferred are a valine residue (V), a leucine residue (L), an isoleucine residue (I) or a glutamine residue (Q), and also most preferred is a glutamine residue (Q).
[0061] A more specific example of the modified fibroin according to the second modality may be a modified fibroin including (2-i) an amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 , or SEQ ID NO: 17; or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17.
[0062] The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 3 is obtained by replacing GQX for all GGX in REP from the amino acid sequence shown in SEQ ID NO: 1 corresponding to naturally occurring fibroin. The amino acid sequence shown in SEQ ID NO: 4 is obtained by deleting the motif (A) _n
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18/59 at each other two positions from the N-terminal side to the C-terminal side of the amino acid sequence shown in SEQ ID NO: 3 and additionally inserting a [(A) _n motif-REP] before the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 10 is obtained by inserting two alanine residues on the C-terminal side of each motif (A) _n of the amino acid sequence shown in SEQ ID NO: 4 and additionally replacing a part of glutamine residues (Q) with a serine residue (S) to delete a portion of amino acids on the N-terminal side in order to have almost the same molecular weight as SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 17 is an amino acid sequence in which a His tag has been added to the C-terminus of a sequence obtained by repeating, 4 times, the region of the 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 10 (meanwhile, several amino acid residues on the C-terminal side of the region are replaced).
[0063] The z / w value in the amino acid sequence shown in 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: 17 is 58.7%, 70.1%, 66.1%, and 70.0%, respectively. In addition, the x / y values in the Giza ratio (to be described later) 1: 1.8 to 1: 11.3 of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 10, and 17 are respectively 15.0%, 15.0%, 93.4%, 92.7%, and 89.3%.
[0064] The modified (2-i) fibroin may consist of an amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17.
[0065] Modified (2-ii) fibroin includes an amino acid sequence having 90% or more sequence identity with the
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19/59 amino acids shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17. Modified (2-ii) fibroin is also a protein including a represented domain sequence by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more. [0066] It is preferred that the modified (2-ii) fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17, ez / w is 50.9% or more in the case where 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 REP is defined as z, and the total number of REP amino acid residues in the domain sequence is defined as w.
[0067] The modified fibroin mentioned above can include a tag sequence in either or both of the N-termination and the C-termination. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
[0068] The tag sequence can be, for example, an affinity tag using specific affinity (binding property, affinity) with another molecule. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property to bind specifically to a metal ion such as nickel, so it can be used to isolate modified fibroin by metal chromatography chelator. A specific example of the tag sequence may be an amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence including His tag).
[0069] In addition, a tag sequence such as glutathione-stransferase (GST) that specifically binds glutathione or a maltose binding protein (MBP) that specifically binds maltose
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20/59 be used.
[0070] Additionally, an "epitope tag" using an antigen-antibody reaction can also be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be linked. Examples of the epitope tag include an HA tag (influenza virus hemagglutinin peptide sequence), a myc tag, and a FLAG tag. Modified fibroin can easily be purified with high specificity using an epitope tag.
[0071] It is also possible to use a tag sequence that can be cleaved with a specific protease. By treating a protein adsorbed through the protease tag sequence, it is also possible to recover the modified fibroin cleaved from the tag sequence.
[0072] A more specific example of the modified fibroin including the tag sequence may be a modified fibroin including (2-iii) an amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 18; or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 18.
[0073] The amino acid sequences shown in SEQ ID NOs: 6, 7, 8, 9, 11, and 18 are amino acid sequences in which an amino acid sequence shown in SEQ ID NO: 5 (including a His tag) is added to the N-terminus of the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 10, and 17, respectively.
[0074] The modified (2-iii) fibroin may consist of an amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 11, or SEQ ID NO: 18.
[0075] Modified (2-iv) fibroin includes a sequence of
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21/59 amino acids having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 18. The modified fibroin from (2 -iv) is also a protein including a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more. [0076] It is preferred that the modified (2-iv) fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 18, ez / w is 50.9% or more in the case where 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 REP is defined as z, and the total number of REP amino acid residues in the domain sequence is defined as w.
[0077] The previously mentioned modified fibroin may include a secretory signal to release the protein produced in the recombinant protein production system out of a host. The sequence of the secretory signal can be properly established depending on the type of the host.
[0078] Fibroin modified according to the third modality has an amino acid sequence whose domain sequence has a reduced content of motif (A) _not compared to naturally occurring fibroin. It can be said that that modified fibroin domain sequence has at least one amino acid sequence corresponding to the deletion of one or a plurality of motifs (A) _n , compared to naturally occurring fibroin. [0079] Fibroin modified according to the third modality can have an amino acid sequence corresponding to 10 to 40% deletion of motif (A) _n of naturally occurring fibroin.
[0080] The modified fibroin according to the third modality can be a modified fibroin whose domain sequence has at least
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22/59 an amino acid sequence corresponding to the deletion of a motif (A) _n by one to three motifs (A) _n from the N-terminal side to the C-terminal side, compared to naturally occurring fibroin.
The modified fibroin according to the third modality can be a modified fibroin whose domain sequence has at least one amino acid sequence corresponding to the repetition of two consecutive motif (A) _n deletions and a motif deletion (A) _{n in} that order from the N-terminal side to the C-terminal side, compared to naturally occurring fibroin.
[0082] The modified fibroin according to the third modality can be a modified fibroin whose domain sequence has at least one amino acid sequence corresponding to the deletion of motif (A) _n at each other two positions from the N-terminal side to the side C-terminal.
[0083] The modified fibroin according to the third modality can be a modified fibroin that has a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and has an amino acid sequence in which x / y is 20% or more, or has an amino acid sequence in which x / y is 50% or more, in the case where the number of amino acid residues in two REPs of adjacent units of [(A) _n motif-REP] is sequentially compared from the N-terminal side to the C-terminal side, and the number of amino acid residues in REP having a smaller number of amino acid residues is defined as 1, the maximum value of the total value of the number of amino acid residues in the two units of adjacent [(A) _n motif-REP] where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is defined as x, and the total number of amino acid residues in the domain sequence is y . This is sufficient as long as the number of alanine residues is 83% or more with respect to the total number of amino acid residues in reason (A) _n , but it is preferably 86% or more, more preferably 90% or more, also more preferably 95% or more, and
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23/59 even more preferably 100% (which means that motif (A) does _not consist only of alanine residues).
[0084] A method for calculating x / y will be described in more detail with reference to FIG. 1. FIG. 1 shows a domain sequence excluding the N-terminal sequence and modified C-terminal sequence of fibroin. This domain sequence has a first REP sequence of motif (A) _n (50 amino acid residues) second REP of motif (A) _n (100 amino acid residues) third REP of motif (A) _n - (10 amino acid residues ) fourth REP of motif (A) _n - (20 amino acid residues) fifth REP of motif (A) _n (30 amino acid residues) -motive (A) _n on the Nterminal side (left side).
[0085] The two adjacent units of [(A) _n motif-REP] 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 motifREP] unit may exist. FIG. 1 shows a pattern 1 (a comparison between first REP and second REP and a comparison between third REP and fourth REP), a pattern 2 (a comparison between first REP and second REP and a fourth comparison between REP and fifth REP), a pattern 3 (a comparison between second REP and third REP and a comparison between fourth REP and fifth REP), and a pattern 4 (a comparison between first REP and second REP). There are other selection methods besides these [0086] Then, for each standard, the number of amino acid residues of each REP in the two adjacent adjacent [(A) _n motif-REP] units is compared. The comparison is performed by obtaining the ratio of the number of amino acid residues of the other REP in the case where a REP having a smaller number of amino acid residues is 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), the ratio of the number of residues of
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24/59 amino acids of the second REP is 100/50 = 2 in the case where the first REP having a smaller number of amino acid residues is 1. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP ( 30 amino acid residues), the ratio of the number of amino acid residues of the fifth REP is 30/20 = 1.5 in the case where the fourth REP having a smaller number of amino acid residues is 1.
[0087] In FIG. 1, a set of units of [(A) _n motif-REP] in which the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 in the case where a REP having a smaller number of amino acid residues is 1 is indicated by a solid line. In the present specification, such a reason is referred to as a Giza reason. A set of units of [(A) _n motif-REP] where the ratio of the number of amino acid residues in the other REP is less than 1.8 or more than 11.3 in the case where a REP having a smaller number of residues of amino acids is 1, is indicated by a dashed line.
[0088] In each standard, the number of all the amino acid residues of two adjacent units of [(A) _n motif-REP] indicated by solid lines (including not only the number of amino acid residues of REP, but also the number of amino acid residues of reason (A) _n ) is combined. Then, the total values thus combined are compared and the total value of the standard whose total value is the maximum (the maximum value of the total value) is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
[0089] Then, x / y (%) can be calculated by dividing x by the number of total amino acid residue y of the domain sequence.
[0090] In fibroin modified according to the third modality, x / y is preferably 50% or more, more preferably 60% or more, also more preferably 65% or more, even more preferably 70% or more, also in form additional preferably
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25/59
75% or more, and in particular preferably 80% or more. The upper limit of x / y is not particularly limited, and it can be 100% or less, for example. In a case where a Giza ratio is 1: 1.9 to 1: 11.3, x / y is preferably 89.6% or more; in a case where a Giza ratio is 1: 1.8 to 1: 3.4, x / y is preferably 77.1% or more; in a case where a Giza ratio is 1: 1.9 to 1: 8.4, x / y is preferably 75.9% or more; and in a case where a Giza ratio is 1: 1.9 to 1: 4.1, x / y is preferably 64.2% or more.
[0091] In a case where the modified fibroin according to the third modality is a modified fibroin in which at least seven of the (A) n motifs that are present in the plural in the domain sequence are composed of only alanine residues, x / y is preferably 46.4% or more, it is more preferably 50% or more, it is even more preferably 55% or more, it is also even more preferably 60% or more, it is also even more preferably 70% or more, and it is particularly 80% or more preferable. The upper limit of x / y is not particularly limited, and can be 100% or less.
[0092] Here, x / y in naturally occurring fibroin will be described. First, as previously described, 663 types of fibroins (415 types of fibroins derived from spiders among them) were extracted confirming fibroins with amino acid sequence information registered in the GenBank NCBI by an exemplified method, x / y was calculated by the mentioned calculation method previously from the naturally occurring amino acid sequences of fibroins that include a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , among all extracted fibroins. FIG. 3 shows the results in the case where the Giza ratio is 1: 1.9 to 1: 4.1.
[0093] In FIG. 3, the horizontal geometric axis represents x / y (%) and the vertical geometric axis represents frequency. As is apparent from FIG. 3, x / y in naturally occurring fibroin is less than 64.2% (more
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26/59 high, 64.14%).
[0094] The modified fibroin according to the third modality can be obtained, for example, from a cloned naturally occurring genetic fibroin sequence, deleting one or a plurality of the sequences that encode the motif (A) _n in such a way that x / y is 64.2% or more. In addition, the modified fibroin including a domain sequence with a reduced motif (A) _{n content} can also be obtained, for example, by designing an amino acid sequence corresponding to the deletion of one or a plurality of motifs (A) n in a manner such that x / y is 64.2% or more of the naturally occurring fibroin amino acid sequence, and chemically synthesizing a nucleic acid that encodes the projected amino acid sequence. In any case, in addition to the modification corresponding to the deletion of motif (A) _n of the naturally occurring fibroin amino acid sequence, modification of the amino acid sequence corresponding to the replacement, deletion, insertion and / or addition of one or a plurality of residues of amino acids can be carried out.
[0095] A more specific example of the modified fibroin according to the third modality can be a modified fibroin including (3-i) an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 , or SEQ ID NO: 17; or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17.
[0096] The modified (3-i) fibroin will be described. The amino acid sequence shown in SEQ ID NO: 2 is obtained by deleting motif (A) _n at each other two positions from the N-terminal side to the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1 corresponding to fibroin naturally occurring and additionally inserting a [(A) _n motifREP] before the C-terminal sequence. The displayed amino acid sequence
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27/59 in SEQ ID NO: 4 is obtained by replacing GQX for all GGX in REP of the amino acid sequence shown in SEQ ID NO: 2. The amino acid sequence shown in SEQ ID NO: 10 is obtained by inserting two alanine residues in the C-terminal side of each motif (A) _n of the amino acid sequence shown in SEQ ID NO: 4 and additionally replacing a portion of glutamine residues (Q) with a serine residue (S) to delete a portion of amino acids on the N-terminal in order to have almost the same molecular weight as SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 17 is an amino acid sequence in which a His tag has been added to the C-terminus of a sequence obtained repeating, 4 times, the region of the 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 10 (meanwhile, several amino acid residues on the C-terminal side of the region are replaced).
[0097] The value of x / y in the ratio Giza 1: 1.8 to 1: 11.3 of the amino acid sequence presented in SEQ ID NO: 1 (corresponding to naturally occurring fibroin) is 15.0%. X / y values in the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 4 are both 93.4%. The x / y value in the amino acid sequence shown in SEQ ID NO: 10 is 92.7%. The x / y value in the amino acid sequence shown in SEQ ID NO: 17 is 89.3%. Z / w values in the amino acid sequences shown in SEQ ID NOs: 1, 2, 4, 10, and 17 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.
[0098] The modified (3-i) fibroin may consist of an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17.
[0099] Modified (3-ii) fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17. Modified (3-ii) fibroin is also a protein
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28/59 including a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.
[00100] The modified fibroin of (3-ii) preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO : 17, and has an amino acid sequence in which x / y is 64.2% or more, in the case where the number of amino acid residues in two REPs of adjacent units of [(A) _n motif-REP] is sequentially compared from the N-terminal side to the C-terminal side, and the number of amino acid residues in REP having a smaller number of amino acid residues is defined as 1, the maximum value of the total value of the number of amino acid residues in the two units of [(A) _n motif-REP] adjacent where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (the Giza ratio of 1: 1.8 to 1: 11.3) is defined as x, and the total number of amino acid residues in the domain sequence is y.
[00101] The modified fibroin mentioned above may include the previously mentioned tag sequence in either or both the N-termination and the C-termination.
[00102] A more specific example of the modified fibroin including the tag sequence may be a modified fibroin including (3-iii) an amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 18; or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 18.
[00103] The amino acid sequences presented in SEQ ID NOs:
6, 7, 8, 9, 11, and 18 are amino acid sequences in which an amino acid sequence shown in SEQ ID NO: 5 (including a His tag) is added to the N-terminus of the amino acid sequences shown in
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SEQ ID NOs: 1, 2, 3, 4, 10, and 17, respectively.
[00104] The modified (3-iii) fibroma may consist of an amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, or SEQ ID NO: 18.
[00105] The modified fibroma of (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 18. The modified (3-iv) fibroma is also a protein including a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.
[00106] The modified (3-iv) fibroin preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO : 18, and has an amino acid sequence in which x / y is 64.2% or more, in the case where the number of amino acid residues in two REPs of adjacent units of [(A) _n motif-REP] is sequentially compared from the N-terminal side to the C-terminal side, and the number of amino acid residues in REP having a smaller number of amino acid residues is defined as 1, the maximum value of the total value of the number of amino acid residues in the two units of [(A) _n motif-REP] adjacent where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is defined as x, and the total number of amino acid residues in the domain sequence is y.
[00107] The previously mentioned modified fibroin may include a secretory signal to release the protein produced in the recombinant protein production system out of a host. The sequence of the secretory signal can be properly established depending on the type of the host.
[00108] Modified fibroin of the fourth modality is a modified fibroin in which the domain sequence has a sequence of
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30/59 amino acids in which the content of glycine residues is reduced in addition to having a reduced content of reasons (A) n compared to naturally occurring fibroin. It can be said that the modified fibroin domain sequence additionally has an amino acid sequence corresponding at least to the replacement of one or a plurality of glycine residues in REP with another amino acid residue, in addition to the deletion of one or one plurality of reasons (A) n, compared to naturally occurring fibroin. That is, the modified fibroin is a modified fibroin which has the resources of the modified fibroin described previously according to the second modality and the modified fibroin described previously according to the third modality. Specific and similar aspects are as described in the modified fibroin according to the second and third modalities.
[00109] A more specific example of the modified fibroin according to the fourth modality may be a modified fibroin including (4-i) an amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17; or (4-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17. Specific aspects of the modified fibroin including the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 17 are as previously described.
<Method for producing modified fibroin>
[00110] Modified fibroin according to the present embodiment can be produced, for example, by expressing the nucleic acid by a nucleic acid sequence encoding the modified fibroin, and a host transformed with an expression vector having one or a plurality of regulatory sequences operably linked to the nucleic acid sequence.
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31/59 [00111] A method for producing a nucleic acid that encodes the modified fibroin is not particularly limited. A nucleic acid can be produced, for example, by a method in which a gene encoding natural fibroin is amplified and cloned by polymerase chain reaction (PCR) or the like, and modified by a genetic engineering method; or a method of chemically synthesizing a nucleic acid. A method for chemically synthesizing a nucleic acid is not particularly limited and, for example, genes can be chemically synthesized by a binding method in which, by PCR or similar, oligonucleotides that are automatically synthesized by AKTA oligopilot plus 10/100 (GE Healthcare Japan Ltd.) or similar, based on the amino acid sequence information of fibroin obtained from the NCBI web database and the like. At this point, in order to facilitate the purification and / or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of a starting codon and a HislO tag to the termination- N from the previous amino acid sequence, can be synthesized.
[00112] The regulatory sequence is a sequence (for example, a promoter sequence, an enhancer, a ribosome binding sequence, or a transcription termination sequence) that controls the expression of a modified fibroin in a host, and can be suitably selected depending on the host type. As a promoter, an inducible promoter that functions in host cells and is capable of inducible expression of modified fibroin can be used. An inducible promoter is a promoter that can control transcription due to the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.
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32/59 [00113] The type of the expression vector such as a plasmid vector, a viral vector, a cosmid vector, a phosmide vector, or an artificial chromosome vector can be appropriately selected depending on the type of the host. Like the expression vector, an expression vector that can replicate autonomously in a host cell or can be incorporated into a host chromosome and that contains a promoter in a position capable of transcribing the nucleic acid encoding the modified fibroin is suitably used .
[00114] Both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used as hosts.
[00115] Examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. Examples of microorganisms that belong to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms that belong to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms that belong to the Serratia genus include Serratia liquefaciens and the like. Examples of microorganisms that belong to the genus Bacillus include Bacillus subtilis and the like. Examples of microorganisms that belong to the genus Microbacterium include Microbacterium ammoniaphilum. Examples of microorganisms that belong to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms that belong to the Corynebacterium genus include Corynebacterium ammoniagenes and the like. Examples of microorganisms that belong to the genus Pseudomonas include Pseudomonas putida and the like.
[00116] In a case where a prokaryote is used as a host, examples of vectors in which a nucleic acid encoding fibroin
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Modified 33/59 is introduced include pBTrp2 (manufactured by Boehringer
Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUBUO, and pNCO2 (Non-Patent Specification Report
Examined Japanese No. 2002-238569) and the like.
[00117] Examples of eukaryotic hosts include yeast and filamentous fungi (mold and the like). Examples of yeasts include a yeast that belongs to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of filamentous fungi include filamentous fungi that belong to the genus Aspergillus, Penicillium, Trichoderma and the like.
[00118] In a case where a eukaryote is used as a host, examples of vectors in which a nucleic acid encoding the modified fibroin are introduced include YEP13 (ATCC37115), YEp24 (ATCC37051) and the like. As a method of introducing an expression vector into the anterior host cell, any method can be used as long as it introduces DNA into the host cell. Examples of the same include a method using calcium ions [Proc. Natl. Acad. Know. USA, 69, 2110 (1972)], an electroporation method, a spheroplast method, a protoplast method, a lithium acetate method, a competent method and the like.
[00119] As a method to express a nucleic acid by a host transformed with an expression vector, secretory production, fusion protein expression, or the like, in addition to direct expression, can be performed according to the method described in Molecular Cloning , 2nd edition.
[00120] The modified fibroin can be produced, for example, by cultivating a host transformed with the expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and then collecting the modified fibroin from the culture medium. The method
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34/59 to cultivate the host in a culture medium can be performed according to a method commonly used to cultivate a host.
[00121] In the case where the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, any of a natural medium and a synthetic medium can be used as a culture medium as long as it contains a carbon source, a source nitrogen, inorganic salts and the like that can be assimilated by the host and it is able to efficiently cultivate the host.
[00122] As the carbon source, any carbon source that can be assimilated by the transformed microorganism can be used. Examples of the carbon source that can be used include carbohydrates such as glucose, fructose, sucrose, and molasses, starch and hydrolyzed starch containing them, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source that can be used include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other compounds containing nitrogen, peptone, meat extract, extract of yeast, corn maceration liquor, casein hydrolyzate, soy cake and soy cake hydrolyzate, several fermented microbial cells and their digested products. As inorganic salts, it is possible to use potassium dihydrogen phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulphate, sodium chloride, ferrous sulphate, manganese sulphate, copper sulphate, and calcium carbonate.
[00123] Culture of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be performed under aerobic conditions such as shaking culture or deep shaking culture. The culture temperature is, for example, 15 ° C to 40 ° C. The culture time is usually 16 hours to 7 days. It is preferable to maintain the pH of the culture medium during culture at 3.0 to 9.0. The pH of the culture medium can be adjusted
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35/59 using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
[00124] In addition, antibiotics such as ampicillin and tetracycline can be added to the culture medium as needed during culture. In the case of cultivating a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer can be added to the medium as needed. For example, in the case of growing a microorganism transformed with an expression vector using a lac promoter, isopropyl-PD-thiogalactopyranoside or the like is used, and in the case of growing a microorganism transformed with an expression vector using a promoter. trp, indole acrylic acid or the like can be added to the medium.
[00125] Isolation and purification of the expressed modified fibroin can be accomplished by a commonly used method. For example, in the case where the modified fibroin is expressed in a dissolved state in cells, the host cells are recovered by centrifugation after completion of the culture, suspended in an aqueous buffer solution, and then broken up using an ultrasound, a French press, a homogenizer Manton-Gaulin, a Dyno-Mill, or the like to obtain a cellless extract. From the supernatant obtained by spinning the cellless extract, a purified preparation can be obtained by a method commonly used to isolate and purify a modified fibroin, that is, a solvent extraction method, a "salinization" precipitation method using ammonium sulfate or the like, a desalting method, a precipitation method using an organic solvent, an ion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Kasei Kogyo Kabushiki Kaisha), a cation exchange chromatography method using a resin such as S-Sepharose FF (Pharmacia Corporation), a
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36/59 hydrophobic chromatography method using a resin such as butyl sepharose or phenyl sepharose, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromato-focusing method, an electrophoresis method such as isoelectric focusing or similar, alone or in combination.
[00126] Furthermore, in the case where the modified fibroin is expressed by the formation of an insoluble substance in the cell, similarly, the host cells are recovered, disrupted and centrifuged to recover the insoluble substance from the modified fibroin as a precipitated fraction. The insoluble substance recovered from the modified fibroin can be solubilized with a protein denaturing agent. After this operation, a purified modified fibroin preparation can be obtained by the same method of isolation and purification as previously described. In the case where 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 by a technique such as spinning, and a purified preparation can be obtained from the culture supernatant using the same isolation and purification method as described above.
<Artificial fibroin fiber [00127] The artificial fibroin fiber according to the present modality is a fiber which is obtained by spinning the modified fibroin described above, and which contains the modified fibroin described above as a main component.
<Method for producing artificial fibroin fiber [00128] The artificial fibroin fiber according to the present embodiment can be produced by a known spinning method. That is, for example, in a case of production of artificial fibroin fibers containing modified fibroin as a main component, first, a dope solution is prepared by adding and dissolving the modified fibroin.
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37/59 produced according to the method described above in a solvent such as dimethyl sulfoxide (DMSO), Ν, Ν-dimethylformamide (DMF), or hexafluorisopronol (HFIP), together with inorganic salt as a dissolution promoter. Then, using this dope solution, spinning is performed by a known spinning method such as wet spinning, dry spinning, wet spinning, or fusion spinning, and thereby fibroin fibers artificial targets can be obtained. Preferred spinning methods include wet spinning or wet spinning.
[00129] FIG. 4 is an explanatory view showing schematically an example of a spinning apparatus for producing artificial fibroin fibers. A spinning apparatus 10 shown in FIG. 4 is an example of a spinning apparatus for wet-dry spinning, and includes an extrusion apparatus 1, an unstretched yarn production apparatus 2, a wet-drawing apparatus 3, and a drying 4.
[00130] A wiring method using the wiring apparatus 10 will be described. First, a dope solution 6 stored in a storage tank 7 is forced through a spinner 9 by a gear pump 8. On the laboratory scale, the dope solution can be filled into a cylinder and forced through the nozzle using a pump. syringe. Then, the extruded dope solution 6 is supplied through an air gap 19 in a coagulation liquid 11 from a coagulation liquid tank 20, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulated body is formed . Then, the fibrous coagulated body is supplied in hot water 12 in a stretch bath 21 and stretched. A stretch ratio is determined by a speed ratio between a roll with feed-through opening 13 and a roll with transfer-through opening 14. After that, the drawn fibrous coagulated body is supplied to the drying apparatus 4 and dried in a line guide 22, and thereby the artificial fibroin fiber as a wound body 5 is
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38/59 obtained. 18a to 18g are wire guides.
[00131] Coagulation liquid 11 can be a solution capable of desolvation, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The clotting liquid 11 can suitably contain water. The temperature of the coagulation liquid 11 is preferably 0 ° C to 30 ° C. In the case where a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the spinner 9, the extrusion rate is preferably 0.2 to 6.0 ml / h and more preferably 1.4 at 4.0 ml / h per hole. The distance that the coagulated protein passes in the coagulation liquid 11 (substantially, the distance from the wire guide 18a to the wire guide 18b) can be any length that allows efficient desolvation and is, for example, 200 to 500 mm. The extraction speed of the unstretched yarn can be, for example, 1 to 20 m / min and preferably 1 to 3 m / min. The residence time in the clotting liquid 11 can be, for example, 0.01 to 3 minutes and preferably 0.05 to 0.15 minutes. In addition, stretching (pre-stretching) can be performed in the coagulation liquid 11.0 coagulation liquid tank 20 can be provided with multiple stages, and the stretching can be performed at each stage or a specific stage, as needed.
[00132] For the stretching performed in a case of obtaining the artificial fibroin fiber, for example, hot stretching by dry route is also employed, in addition to the pre-stretching described previously performed in the coagulation liquid tank 20, and stretching at hot water carried out in the stretch bath 21.
[00133] Hot stretching by wet can be carried out in hot water, in a solution obtained by adding an organic solvent or similar to hot water, or during heating by steam. The temperature can be, for example, 50 ° C to 90 ° C and preferably 75 ° C to 85 ° C. In wet hot drawing, unstretched yarn (or pre-stretched yarn) can
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39/59 be stretched, for example, 1 to 10 times, preferably 2 to 8 times.
[00134] Hot drawing by dry can be carried out using an electric tube oven, a dry thermal plate, or the like. The temperature can be, for example, 140 ° C to 270 ° C and preferably 160 ° C to 230 ° C. In hot dry drawing, unstretched yarn (or pre-stretched yarn) can be stretched, for example, 0.5 to 8 times, preferably 1 to 4 times.
[00135] Hot drawing by wet and hot drawing by dry can be carried out individually, or they can be carried out in multiple stages, or in combination. That is, hot stretch by wet and hot stretch by dry can be performed in an appropriate combination in such a way that the first stretch of the stage is performed by hot stretch by the wet route and the second stretch of the stage is performed by dry hot stretch, or the first stage stretch is performed by wet hot stretch and the second stage stretch is performed by hot wet stretch, and the third stage stretch is additionally performed by stretching hot dry.
[00136] The lower limit of the final stretch ratio is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, or 9 times or more of a stretch ratio of the unstretched yarn (or pre-stretched yarn). The 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, or 10 times or less . <Highly contracted artificial fibroin fiber>
[00137] The artificially contracted fibroin fiber according to the present modality is formed by contraction of the aforementioned artificial fibroin fibers. Artificial fibroin fibers
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40/59 mentioned above can be contracted to a higher percentage of contraction compared to protein fibers known in the related art, and can be contracted to a higher percentage of contraction in milder conditions than those of synthetic fibers known to the related technique.
[00138] Thus, in the highly contracted artificial fibroin fiber according to the present modality, a percentage of contraction defined by the following equation exceeds 7%.
Contraction percentage = {1- (length of artificial fibroin fiber contracted / length of artificial fibroin fiber before being brought into contact with water after spinning)} x 100 (%) [00139] The percentage of contraction of the fibroin fiber highly contracted artificial material according to the present embodiment is preferably 15% or more, it is more preferably more than 25%, it is even more preferably 32% or more, it is also even more preferably 40% or more, it is also even more preferably 48% or more, it is particularly preferably 56% or more, it is even more particularly preferably 64% or more, and it is above all preferably 72% or more. The upper limit of the percentage of contraction is usually 80% or less.
<Method for producing highly contracted artificial fibroin fiber>
[00140] A method for producing a highly contracted artificial fibroin fiber includes a step of contracting an artificial fibroin fiber containing a modified fibroin by placing the artificial fibroin fiber in contact with water below a boiling point (hereinafter referred to as “Contraction stage”).
[00141] In the contraction stage, the artificial fibroin fibers are brought into contact with water below a boiling point (hereinafter also referred to as a "contact stage"). Thereby, fiber
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41/59 of artificial fibroin can be contracted regardless of external force. The contraction without any external force of the artificial fibroin fibers in the contact stage is considered to occur due to the following reasons, for example. In other words, it is considered that, as a reason, it occurs due to a secondary structure and a tertiary structure of artificial fibroin. For example, as another example, it is considered that, in artificial fibroin fibers having residual tension due to stretching or the like in the production process, residual tension is relieved by water infiltration between the fibers or the fibers. In this way, it is considered that a percentage of contraction of the artificial fibroin fiber in the contraction step can be optionally controlled, for example, according to the magnitude of the stretch ratio in the production process of the artificial fibroin fiber described above.
[00142] A water temperature with which the artificial fibroin fibers are brought into contact in the contact step can be less than a boiling point. In this way, the handling and functionality of the contraction process are improved. In addition, from the point of view of sufficiently shortening a contraction time, the value of the lower limit of the water temperature is preferably 10 ° C or more, more preferably 40 ° C or more, and even more preferably 70 ° C or more. The upper limit of the water temperature is preferably 90 ° C or less.
[00143] A method of putting artificial fibroin fibers in contact with water in the contact step is not particularly limited. Examples of methods include a method of immersing artificial fibroin fibers in water; a method of spraying water on artificial fibroin fibers at normal temperature or in a heated steam state and the like; a method of exposing artificial fibroin fibers to a high humidity environment filled with water vapor; and the like. Among these methods, in the contact stage, the method of immersing artificial fibroin fibers in water is
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42/59 preferable because the reduction of the contraction time can be effectively achieved, and simplification of processing equipment can be achieved.
[00144] In the contact stage, when the artificial fibroin fibers are brought into contact with water in a loose state, the artificial fibroin fibers can not only be contracted, but also crimped in a wavy manner. In order to prevent the occurrence of such crimping, for example, the contact step is carried out in a state where the artificial fibroin fibers are not released, placing the artificial fibroin fibers in contact with water below a boiling point, stretching them at the same time (stretching) in a fiber axial direction.
[00145] The contraction step may additionally include, in addition to the contact step, drying the artificial fibroin fibers after being placed in contact with water (hereinafter, it will be referred to as the “drying step”).
[00146] The drying step is a drying step for the artificial fibroin fibers that were submitted to the contact step. Drying can be, for example, natural drying or forced drying using a drying installation. Like drying equipment, any known contact or non-contact drying equipment can be used. In addition, a drying temperature is not particularly limited, as long as it is less than the temperature at which, for example, proteins contained in the artificial fibroin fiber are decomposed, or the artificial fibroin fiber is thermally damaged. In general, the temperature is in the range of 20 ° C to 150 ° C, and the temperature is preferably in the range of 50 ° C to 100 ° C. When the temperature is in this range, the artificial fibroin fibers are dried more quickly and efficiently without thermal damage to the artificial fibroin fibers, or the decomposition of proteins contained in the artificial fibroin fibers. A drying time is set accordingly
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43/59 depending on the drying temperature and the like and, for example, a time during which the influence of supersication on the quality and physical properties of the artificial fibroin fiber can be eliminated as much as possible.
[00147] In the method for producing a highly contracted artificial fibroin fiber according to the present modality, a highly contracted artificial fibroin fiber having a percentage of contraction exceeding 7% can be obtained through the contraction step (the contact step and , if necessary, the drying step). The percentage of contraction of the highly contracted artificial fibroin fiber obtained according to the present embodiment, is preferably 15% or more, it is more preferably more than 25%, it is even more preferably 32% or more, it is also even more preferably 40% or more, it is also even more preferably 48% or more, it is particularly preferably 56% or more, it is even more particularly preferably preferably 64% or more, and it is above all preferably 72% or more. The upper limit of the percentage of contraction is usually 80% or less.
[00148] FIG. 5 is an explanatory view showing schematically an example of a production apparatus for producing highly contracted artificial fibroin fibers. A production apparatus 40 shown in FIG. 5 is configured to include a feed roller 42 for distributing artificial fibroin fibers, a winder 44 for winding highly contracted artificial fibroin fibers 38, a water bath 46 for carrying out the contact step, and a dryer 48 for carrying out the step drying.
[00149] More specifically, the feed roller 42 can be loaded with a product wound from artificial fibroin fibers 36, and the fibers from artificial fibroin 36 are continuously distributed automatically from the product wound from artificial fibroin fibers 36 by rotation of a motor electrical or similar (not shown). Winder 44 can continuously and automatically wind artificial fibroin fibers
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44/59 highly contracted 38 produced through the contact step and the drying step after being fed off the feed roller 42 by the rotation of an electric motor (not shown). A speed of feeding the artificial fibroin fibers 36 by the feed roller 42 and the winding speed of the highly contracted artificial fibroin fibers 38 by the winder 44 can be controlled independently of one another. [00150] The water bath 46 and the dryer 48 are arranged between the feed roller 42 and the winder 44, respectively, on the upstream and downstream side in a feed direction of the artificial fibroin fibers 36. The apparatus production 40 shown in FIG. 5 has transfer rollers 50 and 52 which transfer the artificial fibroin fibers 36 which travel from the feed roller 42 towards the winder 44.
[00151] Water bath 46 has a heater 54, and hot water 47 heated by heater 54 is accommodated in water bath 46. In addition, in water bath 46, a tension roller 56 is installed in a state of being immersed in hot water 47. In this way, the artificial fibroin fibers 36 distributed from the feed roller 42 that travel towards the winder 44 while being immersed in the hot water 47 in a state where they are wound around the tension roller 56 in the water bath 46. An immersion time of the artificial fibroin fibers 36 in the hot water 47 is adequately controlled according to the displacement speed of the artificial fibroin fibers 36.
[00152] The dryer 48 has a pair of hot rollers 58. The pair of hot rollers 58 can be wound with artificial fibroin fibers 36 that are separated from the water bath 46 and move towards the side of the winder 44. In this way, the artificial fibroin fibers 36 immersed in the hot water in the water bath 46 are heated by the pair of hot rollers 58 in the dryer and dried, and then further fed towards the winder 44. [00153] During the production of the artificial fibroin fibers highly
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45/59 contracted 38 using the production apparatus 40 having a structure like this, first, for example, the product wound from the spun artificial fibroin fibers 36 using the spinning apparatus 10 shown in FIG. 4, it is mounted on the feed roller 42. Then the artificial fibroin fibers 36 are continuously extracted from the feed roller 42 and dipped in hot water 47 in the water bath 46. At this moment, for example, the winding speed of the winder 44 becomes less than the feed speed of the feed roller 42. In this way, the crimping generation can be prevented, since the artificial fibroin fibers 36 are contracted when placed in contact with the hot water 47 in a tensioned state so that they are not loose between the feed roller 42 and the winder 44.
[00154] Then, the artificial fibroin fibers 36 contracted in the hot water 47 in the water bath 46 are heated by the pair of hot rollers 58 of the dryer 48. Thereby, the artificial fibroin fibers contracted 36 are dried to form the highly contracted artificial fibroin fibers 38. At this point, by controlling a ratio of the feed speed of the feed roller 42 and the winding speed of the winder 44, it is possible to additionally contract the artificial fibroin fibers 36, or to make their length not be changed. Then, the obtained highly contracted artificial fibroin fibers 38 are wound in the winder 44, and thereby the product wound from the highly contracted artificial fibroin fibers 38 is obtained.
[00155] Instead of the hot roll pair 58, the artificial fibroin fibers 36 can be dried using a drying installation such as a dry thermal plate 64 as shown in FIG. 6. Also in this case, adjusting a relative speed between the feed speed of the feed roller 42 and the winding speed of the winder 44 in the same way as when using the hot roller pair 58 as a
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46/59 drying facility, it is possible to additionally contract the artificial fibroin fibers 36, or to prevent their length from being changed. The drying medium consists of the dry thermal plate 64.
[00156] As previously described, using the production apparatus 40, the highly contracted artificial fibroin fibers 38 can be produced automatically, continuously, and very easily.
[00157] FIG. 6 is an explanatory view showing schematically another example of a production apparatus for producing highly contracted artificial fibroin fibers. FIG. 6 (a) shows a processing device that is included in the production device and that implements the contact step. FIG. 6 (b) shows a drying apparatus which is included in the production apparatus and which implements the drying step. The production apparatus shown in FIG. 6 has a processing apparatus 60 for performing the step of contact with the artificial fibroin fibers 36, and a drying apparatus 62 for drying the fibers of artificial fibroin 36 in which the contact step has been carried out by the processing apparatus 60; and it has a structure in which these devices are independently installed with each other.
[00158] More specifically, the processing apparatus 60 shown in FIG. 6 (a) has a structure in which the dryer 48 is omitted from the production apparatus 40 shown in FIG. 5, and the feed roller 42, the water bath 46, and the winder 44 are arranged in order from the upstream side to the downstream side in a direction of displacement of the artificial fibroin fibers 36. Such processing apparatus 60 is designed to cause the artificial fibroin fibers 36 distributed by the feed roller 42 to be dipped in hot water 47 in the water bath 46 and contracted before being wound by the winder 44. In addition, the structure is configured in such a way that the artificial fibroin fibers 36 contracted in hot water 47 are wound by winder 44.
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47/59 [00159] The drying apparatus 62 shown in FIG. 6 (b) has the feed roller 42, the winder 44, and the dry heat plate 64. The dry heat plate 64 is arranged between the feed roller 42 and the winder 44 in such a way that a dry heating surface 66 is in contact with the artificial fibroin fibers 36 and extends along their displacement direction. In this drying apparatus 62, as previously described, controlling a ratio of the feed speed of the feed roller 42 and the winding speed of the winder 44, it is possible to additionally contract the artificial fibroin fibers 36, or to make their length not to be changed.
[00160] In a case of using the production apparatus having such a structure, for example, the highly contracted artificial fibroin fibers 38 can be produced by first contracting the artificial fibroin fibers 36 by the processing apparatus 60, and then drying the artificial fibroin fibers 36 by the drying apparatus 62.
[00161] The feed roller 42 and the winder 44 can be omitted from the processing apparatus 60 shown in FIG. 6 (a), and the processing apparatus can be configured only with the water bath 46. In a case of using the production apparatus having such a processing apparatus, for example, highly contracted artificial fibroin fibers are produced in a so-called batch system. <Applications for the use of highly contracted artificial fibroin fiber> [00162] The highly contracted artificial fibroin fiber according to the present invention has excellent tactile properties and flexibility because it is contracted at a high percentage of contraction. Therefore, it is suitable as fibers used, for example, for linen and bedding.
[Method for contracting artificial fibroin fiber] [00163] The method for producing an artificial fibroin fiber
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The highly contracted 48/59 of the present invention described above can be perceived as a method for contracting an artificial fibroin fiber, including a step of contracting an artificial fibroin fiber containing a modified fibroin by placing the artificial fibroin fiber in contact with water below a boiling point, where a percentage of contraction defined by the following equation exceeds 7%.
Contraction percentage = {1- (length of the artificial fibroin fiber contracted / length of the artificial fibroin fiber before being put in contact with water after spinning)} x 100 (%).
Examples [00164] In the following, the present invention will be described more specifically on the basis of the examples and the like. However, the present invention is not limited to the following Examples.
[(1) Modified fibroin production (artificial fibroin)] (Nucleic acid synthesis encoding modified fibroin and expression vector construction) [00165] A modified fibroin (PRT399) having the amino acid sequence shown in SEQ ID NO: 7 , a modified fibroin (PRT380) having the amino acid sequence shown in SEQ ID NO: 8, a modified fibroin (PRT410) having the amino acid sequence shown in SEQ ID NO 9, and a modified fibroin (PRT799) having the amino acid sequence presented in SEQ ID NO: 18 have been designed.
[00166] The amino acid sequence shown in SEQ ID NO: 7 is obtained by adding an amino acid sequence (including a His tag) shown in SEQ ID NO: 5 at the N-terminus of an amino acid sequence in which motif (A) _n is deleted at each other two positions from the N-terminal side to the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1 corresponding to naturally occurring fibroin and a [(A) _n motif-REP] is inserted before of the C-terminal sequence.
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49/59 [00167] The amino acid sequence shown in SEQ ID NO: 8 is obtained by adding an amino acid sequence (including a His tag) shown in SEQ ID NO: 5 at the N-terminus of an amino acid sequence in which all GGX in REP of the amino acid sequence shown in SEQ ID NO: 1 corresponding to naturally occurring fibroin are replaced by GQX.
[00168] The amino acid sequence shown in SEQ ID NO: 9 is obtained by adding an amino acid sequence (including a His tag) shown in SEQ ID NO: 5 at the N-terminus of an amino acid sequence in which all GGX in REP of the amino acid sequence shown in SEQ ID NO: 2 are replaced by GQX.
[00169] The amino acid sequence shown in SEQ ID NO: 18 is obtained by adding an amino acid sequence (including a His tag) shown in SEQ ID NO: 5 at the N-terminus of an amino acid sequence in which a His tag has been added to the C-termination of a sequence obtained by repeating, 4 times, the region of the 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 9 (meanwhile, several amino acid residues on the C-terminal side of the region are replaced).
[00170] Nucleic acids encoding the types of types of projected modified fibroin were synthesized respectively. In the nucleic acid, an Ndel site was added to the 5 'end and an EcoRI site was added downstream of the stop codon. These types of nucleic acids have been cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was cleaved by restriction enzyme treatment with Nde and EcoRI, and then recombined in a protein pET-22b (+) expression vector to obtain an expression vector.
(Modified fibroin expression) [00171] Escherichia coli BLR (DE3) was transformed with the vector of
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50/59 pET-22b (+) expression obtained. The transformed Escherichia coli was grown in 2 mL of an LB medium containing ampicillin for 15 hours. The culture solution was added to 100 ml of a seed culture medium (Table 1) containing ampicillin in such a way that the ODôoo was 0.005. The temperature of the culture solution was maintained at 30 ° C and the flask culture was performed (for about 15 hours) until the ODôoo reached 5, thereby obtaining a seed culture solution.
[Table 1]
Seed culture medium
Concentration Reagents (g / L) Glucose 5.0 KH2PO4 4.0 K2HPO4 9.3 Yeast extract 6.0 Ampicillin 0.1
[00172] The seed culture solution was added to a fermentor jar to which 500 ml of production medium (Table 2) were added in such a way that the OD600 was 0.05. The culture was carried out while maintaining the temperature of the culture solution at 37 ° C and keeping the pH constant at 6.9. Additionally, the concentration of oxygen dissolved in the culture solution was maintained at 20% of the saturation concentration of dissolved oxygen.
[Table 2]
Means of production
Reagents Concentration (g / L) Glucose 12.0 KH2PO4 9.0 MgSO ₄ -7H ₂ O 2.4 Yeast extract 15 FeSO ₄ -7H ₂ O 0.04 MnSO ₄ -5H ₂ O 0.04 CaC12-2H ₂ O 0.04 ADEKANOL (LG-295S, Adeka Corporation) 0.1 (mL / L)
[00173] Immediately after glucose in the production medium was completely consumed, a feed solution (455 g / lL of glucose and 120 g / lL of yeast extract) was added at a rate of 1 mL / min. The culture was carried out while maintaining the temperature of the
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51/59 culture at 37 ° C and keeping the pH constant at 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, 1 M isopropyl-P-thiogalactopyranoside (IPTG) was added to the culture solution at a final concentration of 1 mM to introduce the expression of the target modified fibroin. Twenty hours after adding IPTG, the culture solution was centrifuged to recover the bacterial cells. SDS-PAGE was performed using bacterial cells prepared from the culture solution before the addition of IPTG and after the addition of IPTG, and the expression of the target modified fibroin was confirmed by the appearance of a tape of a size of the target modified fibroin depending on the addition of IPTG.
(Purification of modified fibroin) [00174] Bacterial cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCI buffer solution (pH 7.4). The bacterial cells after washing were suspended in a 20 mM TrisHCI buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high pressure homogenizer (available from GEA Niro Soavi SpA). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCI buffer solution (pH 7.4) until high purity. The precipitate after washing was suspended in 8 M guanidine buffer solution (8 M guanidine hydrochloride, 0 mM sodium dihydrogen phosphate, 20 mM NaCl, mM Tris-HC11, pH 7.0) in order to have a concentration of 100 mg / mL, and dissolved by shaking with a shaker at 60 ° C for 30 minutes. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregate protein obtained after dialysis was recovered by spinning. Water was removed from the aggregated protein recovered with a lyophilizer, and the modified fibroin lyophilization powder was obtained.
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52/59 [00175] The degree of purification of the modified target fibroin in the lyophilization powder thus obtained was confirmed by results of image analysis of polyacrylamide gel electrophoresis of the powder using TotalLab (Nonlinear
Dynamics Ltd.). As a result, the purity of each modified fibroin was about 85%.
[(2) Production of artificial fibroin fiber] (Preparation of dope solution) [00176] Dimethyl sulfoxide (DMSO) in which LiCl was dissolved in such a way that a concentration of 4.0% by mass was prepared as a solvent, the lyophilization powder of the modified fibroin was added in such a way that a concentration would be 18% by mass or 24% by mass (refer to Table 3), and the mixture was dissolved for 3 hours using a mixer. After that, insoluble and bubbles were removed, and a modified fibroin solution was obtained.
(Spinning) [00177] The modified fibroin solution obtained was used as a dope solution (undiluted spinning solution), and a spun and stretched artificial fibroin fiber was produced by wet spinning - dry using a wiring that can be considered the wiring apparatus 10 shown in FIG. 4. The spinning apparatus used is an apparatus in which a second unstretched yarn production apparatus (the second bath) is additionally provided between an unstretched yarn production apparatus 2 (the first bath) and a straight drawing apparatus hot water 3 (the third bath) in the spinning apparatus 10 shown in FIG. 1. Conditions for wet spinning - for dry spinning are as follows.
[00178] Diameter of the extrusion nozzle: 0.2 mm
Extrusion speed (amount of discharge): refer to Table 3
Liquid and temperature in the first bath to the third bath:
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53/59 refer to Table 3
Winding speed: refer to Table 3
Total stretch ratio: refer to Table 3
Drying temperature: 60 ° C
Air gap length: refer to Table 3
Petition 870190087335, of 9/5/2019, p. 64/76 [Table 3]
Dope solution Discharge First bath Second bath Third bath Coiling speed (m / min) Total stretch ratio (times) Modified fibroin a Concentration 0 (mass%) Discharge quantity (mg / min) Air gap length (mm) Liquid TemperaturThe(° C) Liquid Temperature (° C) Liquid Temperature (° C) Production example1 PRT799 24 53.8 1 a2 100% methanol -5 100% methanol 16 Water 17 1.45 1 Production example2 2.9 2 Production example3 4.35 3 Production example4 5.8 4 Production example5 18 55.1 1.49 1 Production example6 2.98 2 Production example7 4.47 3 Production example8 5.96 4 Production example9 PRT410 24 51.9 0 -11 14 1.4 1 Production example10 2.8 2 Production example11 4.2 3 Production example12 5.6 4 Production example13 PRT399 51.1 1.38 1 Production example14 2.76 2 Production example15 4.14 3 Production example16 PRT380 47.2 11 1.27 1
54/59
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Production example17 2.54 2 Production example18 3.81 3 Production example19 5.08 4
55/59
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56/59 [(3) Production and evaluation of highly contracted artificial fibroin fiber] (Contraction process) [00179] Highly contracted artificial fibroin fibers were produced by performing, on each artificial fibroin fiber obtained in Production Examples 1 to 19 , a contact step of putting fibers in contact with water below a boiling point (hereinafter referred to as “primary contraction”), or a drying step of drying at room temperature after performing the contact step (hereinafter referred to as “Secondary contraction”).
<Primary contraction>
[00180] A plurality of artificial fibroin fibers each having a length of 30 cm were cut out of the coiled product of the artificial fibroin fibers obtained in Production Examples 1 to 19. The plurality of artificial fibroin fibers were bundled to obtain a bundle of artificial fibroin fiber having a fineness of 150 denier. Each bundle of artificial fibroin fiber was attached with 0.08 g of a lead weight and, in that state, each bundle of artificial fibroin fiber was immersed in water having a temperature shown in Tables 4 to 7 for 10 minutes (step contact details). After that, the length of each bundle of artificial fibroin fiber was measured in water. The measurement of the length of the artificial fibroin fiber bundle in water was performed with the artificial fibroin fiber bundle affixed with a lead weight of 0.08 g in order to eliminate the crimping of the artificial fibroin fiber bundle. Then, a contraction percentage (%) of each artificial fibroin fiber was calculated according to Equation I. In Equation I, L0 indicates the length of the artificial fibroin fiber bundle (here, 30 cm) before being placed in contact with water after spinning, and Lw indicates the length of the bundle of artificial fibroin fiber that underwent primary contraction.
Equation I: percentage of contraction (percentage of primary contraction) =
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57/59 {1- (Lw / LO)} x 100 (%) <Secondary contraction>
[00181] After immersion in water in the primary contraction (the contact step), the bundles of artificial fibroin fiber were removed from the water. The bundles of extracted artificial fibroin fiber were dried at room temperature for 2 hours with a lead weight of 0.08 g attached to this (the drying step). After drying, the length of each bundle of artificial fibroin fiber was measured. Then, a contraction percentage (%) of each artificial fibroin fiber was calculated according to Equation II. In Equation II, L0 indicates the length of the artificial fibroin fiber bundle (here, 30 cm) before being put in contact with water after spinning, and Lwd indicates the length of the artificial fibroin fiber bundle that has undergone primary contraction.
Equation II: percentage of contraction (percentage of secondary contraction) = {I- (Lwd / L0)} x 100 (%) [00182] The results are shown in Tables 4 to 7.
[Table 4] _______________________________________________________________
Artificial fibroin fiber Water temperature below boiling point (° C) Primary contraction (%) Secondary contraction(%) Production example 1 24% by weightPRT799 Xl 20 0.0 7.8 Production example 2 24% by weightPRT799 X2 -1.2 10.3 Production example 3 24% by weightPRT799 X3 7.2 21.2 Production example 4 24% by weightPRT799 X4 13.5 26.3 Production example 6 18% by weightPRT799 X2 -2.3 9.5 Production example 7 18% by weightPRT799 X3 6.0 19.7 Production example 8 18% by weightPRT799 X4 14.3 27.5 Production example 2 24% by weightPRT799 X2 40 -5.3 7.2 Production example 3 24% by weightPRT799 X3 8.7 21.3 Production example 4 24% by weightPRT799 X4 14.5 26.0 Production example 6 18% by weightPRT799 X2 -4.3 7.3
Petition 870190087335, of 9/5/2019, p. 68/76
58/59
Production example 7 18% by weightPRT799 x36.2 18.3 Production example 8 18% by weightPRT799 X4 16.0 28.7 Production example 3 24% by weightPRT799 x3 60 6.8 21.0 Production example 4 24% by weightPRT799 X4 15.0 27.5 Production example 6 18% by weightPRT799 X2 -1.5 10.7 Production example 7 18% by weightPRT799 x3 3.3 18.2 Production example 8 18% by weightPRT799 X4 16.2 29.0
[Table 5]
Artificial fibroin fiber Water temperature below boiling point (° C) Primary contraction (%) Secondary contraction(%) Production example 10 24% by weightPRT410 X2 20 -2.3 8.7 Production example 11 24% by weightPRT410 X3 4.7 16.7 Production example 12 24% by weightPRT410 X4 10.3 22.3 Production example 11 24% by weightPRT410 X3 40 4.7 17.5 Production example 12 24% by weightPRT410 X4 11.5 24.0 Production example 11 24% by weightPRT410 X3 60 2.0 16.5 Production example 12 24% by weightPRT410 X4 1.08 25.0
[Table 6]
Artificial fibroin fiber Water temperature below boiling point (° C) Primary contraction (%) Secondary contraction(%) Production example 13 24% by weightPRT399 xl 20 -3.5 7.6 Production example 14 24% by weightPRT399 X2 3.7 12.5 Production example 15 24% by weightPRT399 x3 7.0 16.8 Production example 14 24% by weightPRT399 X2 40 3.0 12.7 Production example 15 24% by weightPRT399 x3 7.3 16.7 Production example 14 24% by weightPRT399 X2 60 3.3 9.3 Production example 15 24% by weightPRT399 x3 6.8 14.2
[Table 7]
Petition 870190087335, of 9/5/2019, p. 69/76
59/59
Artificial fibroin fiber Water temperature below boiling point (° C) Primary contraction (%) Secondary contraction(%) Production example 16 24% by weightPRT380 Xl 20 -1.1 9.4 Production example 17 24% by weightPRT380 X2 2.7 13.3 Production example 18 24% by weightPRT380 x3 7.0 17.7 Production example 19 24% by weightPRT380 X4 10.0 20.2 Production example 17 24% by weightPRT380 X2 40 3.3 14.2 Production example 18 24% by weightPRT380 x3 7.7 19.0 Production example 19 24% by weightPRT380 X4 12.0 22.0 Production example 17 24% by weightPRT380 X2 60 2.7 14.3 Production example 18 24% by weightPRT380 x3 8.2 20.3 Production example 19 24% by weightPRT380 X4 12.0 23.2
[00183] The highly contracted artificial fibroin fiber of the present invention had a sufficiently high percentage of contraction and was excellent in tactile properties and flexibility. In addition, highly contracted artificial fibroin fiber can be safely produced because it can be manufactured by the contact step of being placed in contact with water below a boiling point, and the drying step of drying the fiber artificial fibroin after the contact step, if necessary.
List of Reference Signs
1: extrusion apparatus, 2: unstretched yarn production apparatus, 3: wet hot drawing apparatus, 4: drying apparatus, 6: dope solution, 10: spinning apparatus, 20: coagulation, 21: stretch bath, 36: artificial fibroin fiber, 38: highly contracted artificial fibroin fiber, 40: production apparatus, 42: feed roller, 44: winder, 46: water bath, 48: dryer, 54: heater, 56: tension roller, 58: hot roller, 60: processing device, 62: drying device, 64: dry heat plate.

权利要求:
Claims (13)
[1]
1. Highly contracted artificial fibroin fiber, characterized by the fact that it comprises:
a modified fibroin, in which the percentage of contraction defined by the following equation exceeds 7%.
contraction percentage = {1- (length of artificial fibroin fiber contracted / length of artificial fibroin fiber before being brought into contact with water after spinning)} x 100 (%)
[2]
2. Highly contracted artificial fibroin fiber according to claim 1, characterized by the fact that the modified fibroin is a modified spider silk fibroin.
[3]
3. Highly contracted artificial fibroin fiber according to claim 1 or 2, characterized by the fact that it is contracted when placed in contact with water below a boiling point.
[4]
4. Highly contracted artificial fibroin fiber according to claim 3, characterized in that the water temperature is 10 ° C to 90 ° C.
[5]
5. Highly contracted artificial fibroin fiber according to claim 3 or 4, characterized in that it is additionally contracted by drying after being placed in contact with water.
[6]
6. Method for producing a highly contracted artificial fibroin fiber, characterized by the fact that it comprises:
a step of contracting an artificial fibroin fiber containing a modified fibroin by placing the artificial fibroin fiber in contact with water below a boiling point, where a percentage of contraction defined by the following equation exceeds 7%.
contraction percentage = {1- (fiber length
Petition 870190087335, of 9/5/2019, p. 71/76
2/3 artificial fibroin contracted / length of artificial fibroin fiber before being brought into contact with water after spinning)} x 100 (%)
[7]
7. Method for producing a highly contracted artificial fibroin fiber according to claim 6, characterized in that the modified fibroin is a modified spider silk fibroin.
[8]
8. Method for producing a highly contracted artificial fibroin fiber according to claim 6 or 7, characterized in that the water temperature is 10 ° C to 90 ° C.
[9]
Method for producing a highly contracted artificial fibroin fiber according to any of claims 6 to 8, characterized in that the step of contracting the artificial fibroin fiber includes additionally drying the artificial fibroin fiber after being brought into contact with the water.
[10]
10. Method for contracting an artificial fibroin fiber, characterized by the fact that it comprises:
a step of contracting an artificial fibroin fiber containing a modified fibroin by placing the artificial fibroin fiber in contact with water below a boiling point, where a percentage of contraction defined by the following equation exceeds 7%.
contraction percentage = {1- (length of artificial fibroin fiber contracted / length of artificial fibroin fiber before being brought into contact with water after spinning)} x 100 (%)
[11]
11. Method for contracting an artificial fibroin fiber according to claim 10, characterized in that the modified fibroin is a modified spider silk fibroin.
[12]
12. Method for contracting an artificial fibroin fiber according to claim 10 or 11, characterized in that the water temperature is 10 ° C to 90 ° C.
Petition 870190087335, of 9/5/2019, p. 72/76
3/3
[13]
13. Method of contracting an artificial fibroin fiber according to any of claims 10 to 12, characterized in that the step of contracting the artificial fibroin fiber includes additionally drying the artificial fibroin fiber after being brought into contact with Water.

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同族专利:

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公开号 | 公开日

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KR20190120384A|2019-10-23|

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US20200031887A1|2020-01-30|

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JP6337252B1|2018-06-06|

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JP2018150637A|2018-09-27|

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EP3594383A4|2020-12-16|

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CA3055888A1|2018-09-13|

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RU2019130386A|2021-04-12|

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RU2757909C2|2021-10-22|

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AU2018232163A1|2019-10-31|

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RU2019130386A3|2021-04-12|

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WO2018164021A1|2018-09-13|

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EP3594383A1|2020-01-15|

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CN110475917A|2019-11-19|

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法律状态:
2021-05-04| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-08-17| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-11-30| B09B| Patent application refused [chapter 9.2 patent gazette]|

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2022-02-15| B09B| Patent application refused [chapter 9.2 patent gazette]|Free format text: MANTIDO O INDEFERIMENTO UMA VEZ QUE NAO FOI APRESENTADO RECURSO DENTRO DO PRAZO LEGAL |

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JP2017046524A|JP6337252B1|2017-03-10|2017-03-10|High shrinkage artificial fibroin fiber, method for producing the same, and method for shrinking artificial fibroin fiber|

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PCT/JP2018/008199|WO2018164021A1|2017-03-10|2018-03-05|Highly contracted synthetic fibroin fiber, production method therefor, and method for contracting synthetic fibroin fiber|

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