Patent Grant
12163259
A computer readable text file, entitled “SequenceListing.txt,” created on or about Mar. 23, 2021 with a file size of about 244 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
The present invention relates to a method for producing a protein spun yarn.
The present inventors proposed a method capable of efficiently producing a protein spun yarn at low cost, by water-crimping protein filaments (Patent Literature 1, unpublished).
Patent Literature 1: Japanese Patent Application No. 2018-15588
However, the present inventors conducted further research, and as a result, it was found that, in a case where spinning was performed after crimping, crimped protein fibers were stretched in a carding process, thus weakening the crimping and reducing interlacing between fibers. As a result, a strength of a spun yarn can be decreased.
An object of the present invention is to provide a method for producing protein spinning capable of securing a stable strength by securing sufficient interlacing between fibers.
The present inventors found that, when producing a protein spun yarn, sufficient interlacing between fibers can be secured, and thus a stable strength of the spun yarn can be secured, by bringing a raw material spun yarn including an uncrimped artificial fibroin fiber containing modified fibroin into contact with an aqueous medium to crimp the artificial fibroin fiber. The present invention is based on this novel finding.
For example, the present invention relates to each of the following inventions.
[1] A method for producing a protein spun yarn, the method including:
[2] The method for producing a protein spun yarn according to [1], in which a dry shrinkage rate of the artificial fibroin fiber, which is defined by the following equation, is higher than 7%:
[3] The method for producing a protein spun yarn according to [1] or [2], in which a wet shrinkage rate of the artificial fibroin fiber, which is defined by the following equation, is 2% or higher:
[4] The method for producing a protein spun yarn according to any one of [1] to [3], in which the modified fibroin is modified spider silk fibroin, and
[5] The method for producing a protein spun yarn according to any one of [1] to [4], in which the aqueous medium used in the crimping step is a liquid or a gas which is at a temperature of 10° C. to 230° C. and contains water.
[6] The method for producing a protein spun yarn according to any one of [1] to [5], in which the crimping step further includes drying after the raw material spun yarn is brought into contact with the aqueous medium.
[7] The method for producing a protein spun yarn according to any one of [1] to [6], in which the aqueous medium used in the crimping step contains a volatile solvent.
According to the method for producing a protein spun yarn of the present invention, a method for producing protein spinning capable of securing a stable strength by securing sufficient interlacing between fibers can be provided.
A method for producing a protein spun yarn according to the present embodiment includes a step (a) of preparing a raw material spun yarn including an uncrimped artificial fibroin fiber containing modified fibroin and a step (b) of bringing the raw material spun yarn into contact with an aqueous medium to crimp the artificial fibroin fiber.
[Step (a)]
(Modified Fibroin)
The modified fibroin according to the present embodiment is a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif. An amino acid sequence (N-terminal sequence or C-terminal sequence) may be further added to any one or both of the N-terminal side and the C-terminal side of the domain sequence of the modified fibroin. The N-terminal sequence and the C-terminal sequence are typically regions not containing repeats of amino acid motifs that are characteristic of fibroin, and consist of about 100 residues of amino acids, but are not limited thereto.
The modified fibroin may be fibroin of which the domain sequence is different from an amino acid sequence of naturally derived fibroin or may be fibroin of which the domain sequence is the same as the amino acid sequence of the naturally derived fibroin. The “naturally derived fibroin” described in the present specification is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A), motif.
As the “modified fibroin”, the amino acid sequence of the naturally derived fibroin may be used as it is, modified fibroin obtained by performing amino acid sequence modification based on the amino acid sequence of the naturally derived fibroin (for example, modified fibroin obtained by performing amino acid sequence modification by modifying a cloned gene sequence for the naturally derived fibroin) may be used, or modified fibroin artificially designed and synthesized independent of the naturally derived fibroin (for example, modified fibroin having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding a designed amino acid sequence) may be used.
The “domain sequence” in the present specification is an amino acid sequence giving rise to a crystalline region characteristic of fibroin (typically corresponds to the (A)n motif in the amino acid sequence) and a non-crystalline region characteristic of fibroin (typically corresponds to REP in the amino acid sequence) and refers to an amino acid sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif. The (A), motif represents an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues therein is 2 to 27. The number of the amino acid residues in the (A)n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. In addition, a ratio of the number of alanine residues to the total number of the amino acid residues in the (A)n motif may be 40% or higher, or may also be 60% or higher, 70% or higher, 80% or higher, 83% or higher, 85% or higher, 86% or higher, 90% or higher, 95% or higher, or 100% (meaning that the (A)n motif only consists of alanine residues). In a case where a plurality of the (A)n motifs are present in the domain sequence, at least 7 of the (A)n motifs may only consist of alanine residues. REP represents an amino acid sequence consisting of 2 to 200 amino acid residues. REP may also be an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. In the case where a plurality of the (A)n motifs are present, the amino acid sequences thereof may be the same or may be different from each other. In a case where a plurality of REP's are present, the amino acid sequences thereof may be the same or may be different from each other.
The modified fibroin according to the present embodiment can be obtained by, for example, performing amino acid sequence modification corresponding to a substitution, a deletion, an insertion, and/or an addition of one of a plurality of amino acid residues with respect to, for example, for a cloned gene sequence derived from the naturally derived fibroin. The substitution, the deletion, the insertion, and/or the addition of an amino acid residue can be performed by a method known to those skilled in the art, such as a site-directed mutagenesis method. Specifically, the substitution, the deletion, the insertion, and/or the addition of an amino acid residue can be performed according to a method described in a literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).
The naturally derived fibroin is a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, and specific examples thereof can include fibroin produced by insects or spiders.
Examples of the fibroin produced by insects can include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama and a hornet silk protein secreted by larvae of Vespa simillima xanthoptera.
More specific examples of the fibroin produced by insects can include the silkworm fibroin L chain (GenBank accession numbers M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
Examples of the fibroin produced by spiders can include spider silk proteins produced by spiders belonging to the genus Araneus, such as Araneus ventricosus, Araneus diadematus, Araneus pinguis, Araneus pentagrammicus, and Araneus nojimai, spiders belonging to the genus Neoscona, such as Neoscona scylla, Neoscona nautica, Neoscona adianta, and Neoscona scylloides, spiders belonging to the genus Pronus, such as Pronous minutus, spiders belonging to the genus Cyrtarachne, such as Cyrtarachne bufo and Cyrtarachne inaequialis, spiders belonging to the genus Gasteracantha, such as Gasteracantha kuhlii and Gasteracantha mammosa, spiders belonging to the genus Ordgarius, such as Ordgarius hobsoni and Ordgarius sexspinosus, spiders belonging to the genus Argiope, such as Argiope amoena, Argiope minuta, and Argiope bruennichi, spiders belonging to the genus Arachnura, such as Arachnura logio, 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 octotuberculata, Cyclosa sedeculata, Cyclosa vallata, and Cyclosa atrata, and spiders belonging to the genus Chorizopes, such as Chorizopes nipponicus, and spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Tetragnatha, such as Tetragnatha praedonia, Tetragnatha maxillosa, Tetragnatha extensa, and Tetragnatha squamata, spiders belonging to the genus Leucauge, such as Leucauge magnifica, Leucauge blanda, and Leucauge subblanda, spiders belonging to the genus 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, Latrodectus hasseltii, Latrodectus geometricus, and Latrodectus tredecimguttatus, and spiders belonging to the genus Euprosthenops. Examples of the spider silk proteins can include dragline silk proteins such as MaSps (MaSp1 and MaSp2) and ADFs (ADF3 and ADF4), MiSps (MiSp1 and MiSp2), and the like.
More specific examples of the spider silk proteins produced by spiders can include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence) and U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus](GenBank accession numbers AAC47011 (amino acid sequence) and U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession numbers AAC04504 (amino acid sequence) and U37520 (base sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence) and EF595246 (base sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence) and AF441245 (base sequence)), major ampullate spidroin 1 [derived from Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence) and AJ973155 (base sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession numbers CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), and the like.
More specific examples of the naturally derived fibroin can further include fibroin of which the sequence information is registered in NCBI GenBank. For example, the fibroin can be verified by extracting, from sequences containing INV as DIVISION, which is one of the sequence information registered in NCBI GenBank, a sequence having a keyword such as spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” described under DEFINITION and a sequence having a specific character string of product described under CDS and a specific character string of TISSUE TYPE described under SOURCE.
The modified fibroin according to the present embodiment may be modified silk fibroin (fibroin obtained by modifying an amino acid sequence of a silk protein produced by silkworms), or may be modified spider silk fibroin (fibroin obtained by modifying an amino acid sequence of a spider silk protein produced by spiders). As the modified fibroin, the modified spider silk fibroin is preferred.
Specific examples of the modified fibroin can include modified fibroin derived from a spigot dragline silk protein produced in a major ampullate gland of a spider (first modified fibroin), modified fibroin having a domain sequence in which a content of glycine residues is reduced (second modified fibroin), modified fibroin having a domain sequence in which a content of the (A)n motifs is reduced (third modified fibroin), modified fibroin in which the contents of glycine residues and the (A)n motifs are reduced (fourth modified fibroin), modified fibroin having a domain sequence containing a region in which a hydropathy index is locally high (fifth modified fibroin), and modified fibroin having a domain sequence in which a content of glutamine residues is reduced (sixth modified fibroin).
Examples of the first modified fibroin can include a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m. The number of amino acid residues in the (A)n motif in the first modified fibroin is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, still more preferably an integer of 10 to 20, still even more preferably an integer of 4 to 16, particularly preferably an integer of 8 to 16, and most preferably an integer of 10 to 16. The number of amino acid residues constituting REP in Formula 1 in the first modified fibroin is preferably 10 to 200 residues, more preferably 10 to 150 residues, even more preferably 20 to 100 residues, and still more preferably 20 to 75 residues. A total number of a glycine residue, a serine residue, and an alanine residue contained in the amino acid sequence represented by Formula 1: [(A)n motif-REP]m in the first modified fibroin 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.
The first modified fibroin may be a polypeptide which contains a unit of an amino acid sequence represented by Formula 1: [(A)n motif-REP]m and of which the C-terminal sequence is an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3 or an amino acid sequence having an identity of 90% or higher with an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3.
The amino acid sequence set forth in SEQ ID NO: 1 is the same as an amino acid sequence consisting of 50 amino acid residues at the C-terminus of the amino acid sequence of ADF3 (GI: 1263287, NCBI), the amino acid sequence set forth in SEQ ID NO: 2 is the same as an amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence set forth in SEQ ID NO: 3 is the same as an amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence set forth in SEQ ID NO: 1.
More specific examples of the first modified fibroin can include modified fibroin having (1-i) an amino acid sequence set forth in SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 4. It is preferable that the sequence identity is 95% or higher.
The amino acid sequence set forth in SEQ ID NO: 4 is obtained by causing mutations so that, in an amino acid sequence of ADF3 to which an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, a His10-tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site is added at the N-terminus, the 1st to 13th repeat regions are increased to be nearly doubled, and the translation is terminated at the 1,154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 4 is the same as the amino acid sequence set forth in SEQ ID NO: 3.
The modified fibroin of (1-i) may consist of the amino acid sequence set forth in SEQ ID NO: 4.
The domain sequence of the second modified fibroin has an amino acid sequence in which the content of glycine residues is reduced compared to the naturally derived fibroin. The second modified fibroin can be defined as fibroin having an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of glycine residues in REP are substituted by other amino acid residues, compared to the naturally derived fibroin.
The domain sequence of the second modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which at least one glycine residue in one or a plurality of motif sequences is substituted by another amino acid residue, compared to the naturally derived fibroin, the motif sequence being at least one motif sequence selected from GGX and GPGXX (here, G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP.
In the second modified fibroin, a ratio of the above-described motif sequence in which a glycine residue is substituted by another amino acid residue to the total motif sequences may be 10% or higher.
The second modified fibroin has a domain sequence represented by Formula 1: [(A)n motif-REP]m, and in a case where a total number of amino acid residues in amino acid sequences consisting of XGX (here, X represents an amino acid residue other than glycine) contained in all REP's in the sequences in the domain sequence excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by z, and a total number of amino acid residues in the domain sequence excluding the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by w, the second modified fibroin may have an amino acid sequence in which z/w is 30% or higher, 40% or higher, 50% or higher, or 50.9% or higher. The number of alanine residues with respect to the total number of amino acid residues in the (A)n motif may be 83% or higher, preferably 86% or higher, more preferably 90% or higher, even more preferably 95% or higher, and still more preferably 100% (meaning that the (A)n motif only consists of alanine residues).
It is preferable that a content ratio of the amino acid sequence consisting of XGX in the second modified fibroin is increased by substituting one glycine residue in the GGX motif with another amino acid residue. A content ratio of the amino acid sequence consisting of GGX in the domain sequence of the second modified fibroin is preferably 30% or lower, more preferably 20% or lower, even more preferably 10% or lower, still more preferably 6% or lower, still even more preferably 4% or lower, and particularly preferably 2% or lower. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated using the same method as the method for calculating the content ratio of the amino acid sequence consisting of XGX (z/w) below.
The method for calculating z/w will be described in further detail. First, the amino acid sequence consisting of XGX is extracted from all REP's contained in a domain sequence of fibroin (modified fibroin or naturally derived fibroin), which has a domain sequence represented by Formula 1: [(A)n motif-REP]m, excluding a sequence from the (A), motif located closest to the C-terminal side to the C-terminus of the domain sequence. A total number of amino acid residues constituting XGX is denoted by z. For example, in a case where 50 amino acid sequences consisting of XGX are extracted (without overlaps), z is 50×3=150. Furthermore, in a case where X belonging to two XGX's is present, as in the case of, for example, an amino acid sequence consisting of XGXGX (X in the center), z is calculated by deducting the overlapping amino acid residue (in the case of XGXGX, the number of amino acid residues is 5). w is a total number of amino acid residues in the domain sequence excluding the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence. For example, in a case of the domain sequence shown in
Here, z/w in the naturally derived fibroin will be described. First, fibroin of which the amino acid sequence information is registered in NCBI GenBank was verified as described above using the method exemplified above, and as a result, 663 types of fibroin (among these, 415 types were fibroin derived from spiders) were extracted. Among all extracted fibroin, values of z/w were calculated, using the calculation method described above, from amino acid sequences of naturally derived fibroin which contained domain sequences represented by Formula 1: [(A)n motif-REP]m and in which the content ratios of the amino acid sequences consisting of GGX in the fibroins were 6% or lower. The results are shown in
z/w in the second modified fibroin is preferably 50.9% or higher, more preferably 56.1% or higher, even more preferably 58.7% or higher, still more preferably 70% or higher, and still even more preferably 80% or higher. The upper limit of z/w is not particularly limited, and may be, for example, 95% or lower.
The second modified fibroin can be obtained by, for example, performing modification so that at least a part of base sequences encoding glycine residues in a cloned gene sequence for the naturally derived fibroin are substituted so as to encode other amino acid residues. In this case, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, and the substitution may be performed so that z/w is 50.9% or higher. It is also possible to obtain the second modified fibroin by, for example, designing an amino acid sequence satisfying the above aspect from the amino acid sequence of the naturally derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to a substitution of a glycine residue in REP in the amino acid sequence of the naturally derived fibroin with another amino acid residue, further amino acid sequence modification may be performed, which corresponds to a substitution, a deletion, an insertion, and/or an addition of one or a plurality of amino acid residues.
Another amino acid residue above is not particularly limited as long as it is an amino acid residue other than a glycine residue, and the amino acid residue is preferably a hydrophobic amino acid residue such as a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a methionine (M) residue, a proline (P) residue, a phenylalanine (F) residue, and a tryptophan (W) residue, and a hydrophilic amino acid residue such as a glutamine (Q) residue, an asparagine (N) residue, a serine (S) residue, a lysine (K) residue, and a glutamic acid (E) residue, more preferably a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a phenylalanine (F) residue, and a glutamine (Q) residue, and even more preferably a glutamine (Q) residue.
More specific examples of the second modified fibroin can include modified fibroin having (2-i) an amino acid sequence set forth in SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met-PRT799) or (2-ii) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (2-i) will be described. The amino acid sequence set forth in SEQ ID NO: 6 is obtained by substituting all GGX's in REP in an amino acid sequence set forth in SEQ ID NO: 10 (Met-PRT313), which corresponds to the naturally derived fibroin, with GQX's. The amino acid sequence set forth in SEQ ID NO: 7 is obtained from the amino acid sequence set forth in SEQ ID NO: 6, by deleting an (A)n motif at every other two positions from the N-terminal side to the C-terminal side and inserting one [(A)n motif-REP] before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 8 is obtained by inserting two alanine residues on the C-terminal side of each (A)n motif in the amino acid sequence forth in SEQ ID NO: 7, substituting a part of glutamine (Q) residues with serine (S) residues, and deleting a part of amino acids on the C-terminal side so that the molecular weight thereof is about the same as the molecular weight of the amino acid sequence set forth in SEQ ID NO: 7. The amino acid sequence set forth in SEQ ID NO: 9 is obtained by adding a predetermined hinge sequence and His-tag sequence to the C-terminus of a sequence in which a region of 20 domain sequences (here, several amino acid residues on the C-terminal side of the region are substituted) existing in the amino acid sequence set forth in SEQ ID NO: 7 are repeated four times.
A value of z/w in the amino acid sequence set forth in SEQ ID NO: 10 (corresponding to naturally derived fibroin) is 46.8%. Values of z/w in the amino acid sequence set forth in SEQ ID NO: 6, the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, and the amino acid sequence set forth in SEQ ID NO: 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. Furthermore, values of x/y in the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 at a Giza ratio (to be described later) of 1:1.8 to 11.3 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.
The modified fibroin of (2-i) may consist of the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (2-ii) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m. It is preferable that the sequence identity is 95% or higher.
The modified fibroin of (2-ii) has a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and in a case where a total number of amino acid residues in amino acid sequences consisting of XGX (here, X represents an amino acid residue other than glycine) which are contained in REP is denoted by z, and a total number of amino acid residues in REP's in the domain sequence is denoted by w, z/w is preferably 50.9% or higher.
The second modified fibroin may have a tag sequence at one or both of the N-terminus and the C-terminus thereof. By having a tag sequence, isolation, immobilization, detection, visualization, and the like of the modified fibroin become possible.
Examples of the tag sequence can include an affinity tag using specific affinity (binding properties or affinity) to another molecule. Specific examples of the affinity tag can include a histidine tag (His-tag). The His-tag is a short peptide in which about 4 to 10 histidine residues are lined up and can be used for isolating modified fibroin by chelating metal chromatography, since it has a property of specifically binding to metal ions such as nickel. Specific examples of the tag sequence can include an amino acid sequence set forth in SEQ ID NO: 11 (an amino acid sequence having a His-tag sequence and a hinge sequence).
Furthermore, tag sequences such as a glutathione S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can also be used.
In addition, an “epitope tag” using an antigen-antibody reaction can also be used. By adding a peptide showing antigenicity (epitope) as a tag sequence, an antibody to the epitope can bind to the modified fibroin. Examples of the epitope tag can include an HA (a peptide sequence of influenza virus hemagglutinin) tag, a myc tag, a FLAG tag, and the like. The use of the epitope tag allows purification of the modified fibroin to be easily performed with high specificity.
In addition, a tag sequence that can be separated by a specific protease can also be used. By treating a protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence is separated can be recovered.
More specific examples of the modified fibroin having a tag sequence can include modified fibroin having (2-iii) an amino acid sequence set forth in SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) or (2-iv) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
The amino acid sequences set forth in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (which has a His-tag sequence and a hinge sequence) to the N-termini of the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.
The modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
The modified fibroin of (2-iv) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m. It is preferable that the sequence identity is 95% or higher.
The modified fibroin of (2-iv) has a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and in a case where a total number of amino acid residues in amino acid sequences consisting of XGX (here, X represents an amino acid residue other than glycine) which are contained in REP is denoted by z, and a total number of amino acid residues in REP's in the domain sequence is denoted by w, z/w is preferably 50.9% or higher.
The second modified fibroin may have a secretory signal for releasing a protein produced in a recombinant protein production system to the outside of a host. A sequence of the secretory signal can be suitably set according to the type of the host.
The domain sequence of the third modified fibroin has an amino acid sequence in which the content of the (A)n motifs is reduced compared to the naturally derived fibroin. The domain sequence of the third modified fibroin can be defined as a domain sequence having an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of the (A)n motifs are deleted, compared to the naturally derived fibroin.
The third modified fibroin may have an amino acid sequence corresponding to an amino acid sequence obtained by deleting 10% to 40% of the (A)n motifs in the naturally derived fibroin.
The domain sequence of the third modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which at least one (A)n motif in every one to three (A)n motifs is deleted from the N-terminal side to the C-terminal side, compared to the naturally derived fibroin.
The domain sequence of the third modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which, at least, a deletion of two consecutive (A)n motifs and a deletion of one (A)n motif are repeated in this order from the N-terminal side to the C-terminal side, compared to the naturally derived fibroin.
The domain sequence of the third modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which an (A)n motif is deleted at at least every other two positions from the N-terminal side to the C-terminal side.
The third modified fibroin has a domain sequence represented by Formula 1: [(A)n motif-REP]m, and in a case of sequentially comparing the numbers of amino acid residues in REP's of two adjacent [(A)n motif-REP] units from the N-terminal side to the C-terminal side and adding up the numbers of amino acid residues in two adjacent [(A), motif-REP] units in which, when the number of amino acid residues in REP having a smaller number of amino acid residues is set to 1, the proportion of the number of the amino acid residues in the other REP is 1.8 to 11.3, so that the maximum value of the sum is denoted by x, and the total number of amino acid residues in the domain sequence is denoted by y, the third modified fibroin may have an amino acid sequence in which x/y is 20% or higher, 30% or higher, 40% or higher, or 50% or higher. The number of alanine residues with respect to the total number of amino acid residues in the (A)n motif may be 83% or higher, preferably 86% or higher, more preferably 90% or higher, even more preferably 95% or higher, and still more preferably 100% (meaning that the (A)n motif only consists of alanine residues).
The method for calculating x/y will be described in further detail while referring to
Two adjacent [(A)n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side without overlaps. In this case, a [(A)n motif-REP] unit that has not been selected may be present.
Next, in each pattern, the numbers of amino acid residues in the REP's of the selected two adjacent [(A)n motif-REP] units are compared with each other. The comparison is performed by, setting the smaller number of amino acid residues to 1, and calculating the proportion of the number of amino acid residues in the other REP therefrom. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the number of amino acid residues in the first REP which is smaller is set to 1, the proportion of the number of amino acid residues in the second REP is 100/50=2. In the same manner, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the number of amino acid residues in the fourth REP which is smaller is set to 1, the proportion of the number of amino acid residues in the fifth REP is 30/20=1.5.
In
In each pattern, all of the numbers of amino acid residues in the two adjacent [(A)n motif-REP] units shown as the solid lines are added up (the numbers of amino acid residues in not only REP, but also in the (A)n motifs are added). The values of the sums are compared with each other, and the value of the sum in a pattern in which the value of the sum is the largest (maximum value of the sum) is denoted by x. In the example illustrated in
Next, x/y (%) can be calculated by dividing x by y, which is the total number of amino acid residues in the domain sequence.
x/y in the third modified fibroin is preferably 50% or higher, more preferably 60% or higher, even more preferably 65% or higher, still more preferably 70% or higher, still even more preferably 75% or higher, and particularly preferably 80% or higher. The upper limit of x/y is not particularly limited, and may be, for example, 100% or lower. In a case where the Giza ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or higher, in a case where the Giza ratio is 1:1.8 to 3.4, x/y is preferably 77.1% or higher, in a case where the Giza ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or higher, and in a case where the Giza ratio is 1:1.9 to 4.1, x/y is preferably 64.2% or higher.
In a case where the third modified fibroin is modified fibroin in which at least 7 of the plurality of (A), motifs present in the domain sequence only consist of alanine residues, x/y is preferably 46.4% or higher, more preferably 50% or higher, even more preferably 55% or higher, still more preferably 60% or higher, still even more preferably 70% or higher, and particularly preferably 80% or higher. The upper limit of x/y is not particularly limited and may be 100% or lower.
Here, x/y in the naturally derived fibroin will be described. First, fibroin of which the amino acid sequence information is registered in NCBI GenBank was verified as described above using the method exemplified above, and as a result, 663 types of fibroin (among these, 415 types were fibroin derived from spiders) were extracted. Among all extracted fibroin, values of x/y were calculated, using the calculation method described above, from amino acid sequences of naturally derived fibroin consisting of domain sequences represented by Formula 1: [(A)n motif-REP]m. The results in a case where the Giza ratio was 1:1.9 to 4.1 are shown in
The horizontal axis in
The third modified fibroin can be obtained by, for example, deleting one or a plurality of sequences encoding the (A)n motif from a cloned gene sequence for the naturally derived fibroin so that x/y is 64.2% or higher. It is also possible to obtain the third modified fibroin by, for example, designing an amino acid sequence corresponding to an amino acid sequence obtained by deleting one or a plurality of (A)n motifs from the amino acid sequence of the naturally derived fibroin so that x/y is 64.2% or higher and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to deletion of the (A)n motif from the amino acid sequence of the naturally derived fibroin, further amino acid sequence modification may be performed, which corresponds to a substitution, a deletion, an insertion, and/or an addition of one or a plurality of amino acid residues.
More specific examples of the third modified fibroin can include modified fibroin having (3-i) an amino acid sequence set forth in SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met-PRT799) or (3-ii) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (3-i) will be described. The amino acid sequence set forth in SEQ ID NO: 17 is obtained from the amino acid sequence set forth in SEQ ID NO: 10 (Met-PRT313) that corresponds to the naturally derived fibroin, by deleting an (A)n motif at every other two positions from the N-terminal side to the C-terminal side and inserting one [(A)n motif-REP] before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is the same as that described for the second modified fibroin.
A value of x/y in the amino acid sequence set forth in SEQ ID NO: 10 (corresponding to the naturally derived fibroin) at a Giza ratio of 1:1.8 to 11.3 is 15.0%. Values of x/y in the amino acid sequence set forth in SEQ ID NO: 17 and the amino acid sequence set forth in SEQ ID NO: 7 are both 93.4%. A value of x/y in the amino acid sequence set forth in SEQ ID NO: 8 is 92.7%. A value of x/y in the amino acid sequence set forth in SEQ ID NO: 9 is 89.8%. Values of z/w in the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.
The modified fibroin of (3-i) may consist of the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (3-ii) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m. It is preferable that the sequence identity is 95% or higher.
The modified fibroin of (3-ii) has a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and in a case of sequentially comparing the numbers of amino acid residues in REP's of two adjacent [(A)n motif-REP] units from the N-terminal side to the C-terminal side and adding up the numbers of amino acid residues in two adjacent [(A)n motif-REP] units in which, when the number of amino acid residues in REP having a smaller number of amino acid residues is set to 1, the proportion of the number of the amino acid residues in the other REP is 1.8 to 11.3 (a Giza ratio is 1:1.8 to 11.3), so that the maximum value of the sum is denoted by x, and the total number of amino acid residues in the domain sequence is denoted by y, x/y is preferably 64.2% or higher.
The third modified fibroin may have the tag sequence described above at one or both of the N-terminus and the C-terminus thereof.
More specific examples of the modified fibroin having a tag sequence can include modified fibroin having (3-iii) an amino acid sequence set forth in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) or (3-iv) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
The amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (which has a His-tag sequence and a hinge sequence) to the N-termini of the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.
The modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
The modified fibroin of (3-iv) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m. It is preferable that the sequence identity is 95% or higher.
The modified fibroin of (3-iv) has a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and in a case of sequentially comparing the numbers of amino acid residues in REP's of two adjacent [(A)n motif-REP] units from the N-terminal side to the C-terminal side and adding up the numbers of amino acid residues in two adjacent [(A)n motif-REP] units in which, when the number of amino acid residues in REP having a smaller number of amino acid residues is set to 1, the proportion of the number of the amino acid residues in the other REP is 1.8 to 11.3, so that the maximum value of the sum is denoted by x, and the total number of amino acid residues in the domain sequence is denoted by y, x/y is preferably 64.2% or higher.
The third modified fibroin may have a secretory signal for releasing a protein produced in a recombinant protein production system to the outside of a host. A sequence of the secretory signal can be suitably set according to the type of the host.
The domain sequence of the fourth modified fibroin has an amino acid sequence having a reduced content of glycine residues, as well as a reduced content of the (A)n motifs, compared to the naturally derived fibroin. The domain sequence of the fourth modified fibroin can be defined as a domain sequence having an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of the (A)n motifs are deleted, and at least one or a plurality of glycine residues in REP are substituted by other amino acid residues, compared to the naturally derived fibroin. That is, the fourth modified fibroin is modified fibroin having characteristics of both the second modified fibroin and the third modified fibroin described above. Specific aspects and the like are the same as those described for the second modified fibroin and the third modified fibroin.
More specific examples of the fourth modified fibroin can include modified fibroin having (4-i) an amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) or (4-ii) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. Specific aspects of the modified fibroin having the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 are as described above.
The domain sequence of the fifth modified fibroin may have an amino acid sequence containing a region in which a hydropathy index is locally high, which corresponds to an amino acid sequence in which one or a plurality of amino acid residues in REP are substituted by amino acid residues having a high hydropathy index, and/or an amino acid sequence in which one or a plurality of amino acid residues having a high hydropathy index are inserted into REP, compared to the naturally derived fibroin.
It is preferable that the region in which a hydropathy index is locally high consists of 2 to 4 consecutive amino acid residues.
The amino acid residue having a high hydropathy index described above is more preferably an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A).
In addition to the modification corresponding to a substitution of one or a plurality of amino acid residues in REP with amino acid residues having a high hydropathy index and/or an insertion of one or a plurality of amino acid residues having a high hydropathy index into REP, compared to the naturally derived fibroin, further amino acid sequence modification may be performed on the fifth modified fibroin, which corresponds to a substitution, a deletion, an insertion, and/or an addition of one or a plurality of amino acid residues, compared to the naturally derived fibroin.
The fifth modified fibroin can be obtained from, for example, a cloned gene sequence for the naturally derived fibroin by substituting one or a plurality of hydrophilic amino acid residues (for example, amino acid residues having a negative value of hydropathy index) in REP with hydrophobic amino acid residues (for example, amino acid residues having a positive value of hydropathy index) and/or by inserting one or a plurality of hydrophobic amino acid residues into REP. It is also possible to obtain the fifth modified fibroin by, for example, designing an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of hydrophilic amino acid residues in REP in the amino acid sequence of the naturally derived fibroin are substituted by hydrophobic amino acid residues and/or an amino acid sequence in which one or a plurality of hydrophobic amino acid residues are inserted into REP in the amino acid sequence of the naturally derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to a substitution of one or a plurality of hydrophilic amino acid residues in REP in the amino acid sequence of the naturally derived fibroin with hydrophobic amino acid residues and/or an insertion of one or a plurality of hydrophobic amino acid residues into REP in the amino acid sequence of the naturally derived fibroin, further amino acid sequence modification may be performed, which corresponds to a substitution, a deletion, an insertion, and/or an addition of one or a plurality of amino acid residues.
The fifth modified fibroin has a domain sequence represented by Formula 1: [(A)n motif-REP]m, and in a case where a total number of amino acid residues contained in regions in which an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or higher in all REP's contained in the domain sequence excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by p, and a total number of amino acid residues contained in the domain sequence excluding the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by q, the fifth modified fibroin may have an amino acid sequence in which p/q is 6.2% or higher.
As the hydropathy index of an amino acid residue, a known index (Hydropathy index: Kyte J and Doolittle R (1982) “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. 105-132) is used. Specifically, a hydropathy index (hereinafter, also referred to as “HI”) of each amino acid is indicated in the following Table 1.
The method for calculating p/q will be described in further detail. In the calculation, a domain sequence represented by Formula 1: [(A)n motif-REP]m is used, excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence (hereinafter, referred to as “sequence A”). First, average values of hydropathy indices of four consecutive amino acid residues in all REP's contained in the sequence A are calculated. The average value of hydropathy indices is calculated by dividing a sum of HI's of all amino acid residues contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of hydropathy indices is calculated for every four consecutive amino acid residues (each amino acid residue is used in the calculation of an average value one to four times). Next, regions in which the average value of hydropathy indices of four consecutive amino acid residues is 2.6 or higher are specified. Even in a case where a certain amino acid residue belongs to a plurality of sets of “four consecutive amino acid residues of which the average value of hydropathy indices is 2.6 or higher”, the amino acid residue is contained in the region as one amino acid residue. A total number of amino acid residues contained in the region is p. Furthermore, a total number of amino acid residues contained in the sequence A is q.
For example, in a case where “four consecutive amino acid residues of which the average value of hydropathy indices is 2.6 or higher” are extracted at 20 locations (without overlaps), 20 sets of four consecutive amino acid residues (without overlaps) are contained in the regions in which the average value of hydropathy indices of four consecutive amino acid residues is 2.6 or higher, and p is 20×4=80. Furthermore, in a case where, for example, only one amino acid residue overlaps within two sets of “four consecutive amino acid residues of which the average value of hydropathy indices is 2.6 or higher”, the region in which the average value of hydropathy indices of four consecutive amino acid residues is 2.6 or higher contains seven amino acid residues (p=2×4-1=7. “−1” is a deduction of the overlapping amino acid residue). For example, in a case of the domain sequence shown in
p/q in the fifth modified fibroin is preferably 6.2% or higher, more preferably 7% or higher, even more preferably 10% or higher, still more preferably 20% or higher, and still even more preferably 30% or higher. The upper limit of p/q is not particularly limited, and may be, for example, 45% or lower.
The fifth modified fibroin can be obtained by, for example, modifying a cloned amino acid sequence of the naturally derived fibroin into an amino acid sequence containing a region in which a hydropathy index is locally high by substituting one or a plurality of hydrophilic amino acid residues (for example, amino acid residues having a negative value of hydropathy index) in REP with hydrophobic amino acid residues (for example, amino acid residues having a positive value of hydropathy index) and/or by inserting one or a plurality of hydrophobic amino acid residues into REP, so that the condition of p/q is satisfied. It is also possible to obtain the fifth modified fibroin by, for example, designing an amino acid sequence satisfying the condition of p/q from the amino acid sequence of the naturally derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to a substitution of one or a plurality of amino acid residues in REP with amino acid residues having a high hydropathy index and/or an insertion of one or a plurality of amino acid residues having a high hydropathy index into REP, compared to the naturally derived fibroin, further modification may be performed, which corresponds to a substitution, a deletion, an insertion, and/or an addition of one or a plurality of amino acid residues.
The amino acid residue having a high hydropathy index is not particularly limited, and is preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A), and more preferably valine (V), leucine (L), and isoleucine (I).
More specific examples of the fifth modified fibroin can include modified fibroin having (5-i) an amino acid sequence set forth in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666) or (5-ii) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
The modified fibroin of (5-i) will be described. The amino acid sequence set forth in SEQ ID NO: 19 is obtained by inserting amino acid sequences each consisting of three amino acid residues (VLI) at two positions for every other REP in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410) excluding the terminal domain sequence on the C-terminal side, substituting a part of glutamine (Q) residues with serine (S) residues, and deleting a part of amino acids on the C-terminal side. The amino acid sequence set forth in SEQ ID NO: 20 is obtained by inserting amino acid sequences each consisting of three amino acid residues (VLI) at one position for every other REP in the amino acid sequence set forth in SEQ ID NO: 8 (Met-PRT525). The amino acid sequence set forth in SEQ ID NO: 21 is obtained by inserting amino acid sequences each consisting of three amino acid residues (VLI) at two positions for every other REP in the amino acid sequence set forth in SEQ ID NO: 8.
The modified fibroin of (5-i) may consist of the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
The modified fibroin of (5-ii) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m. It is preferable that the sequence identity is 95% or higher.
The modified fibroin of (5-ii) has a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and in a case where a total number of amino acid residues contained in regions in which an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or higher in all REP's contained in the domain sequence excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by p, and a total number of amino acid residues contained in the domain sequence excluding the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by q, p/q is preferably 6.2% or higher.
The fifth modified fibroin may have a tag sequence at one or both of the N-terminus and the C-terminus thereof.
More specific examples of the modified fibroin having a tag sequence can include modified fibroin having (5-iii) an amino acid sequence set forth in SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666) or (5-iv) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
The amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 are obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (which has a His-tag sequence and a hinge sequence) to the N-termini of the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.
The modified fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
The modified fibroin of (5-iv) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m. It is preferable that the sequence identity is 95% or higher.
The modified fibroin of (5-iv) has a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and in a case where a total number of amino acid residues contained in regions in which an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or higher in all REP's contained in the domain sequence excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by p, and a total number of amino acid residues contained in the domain sequence excluding the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by q, p/q is preferably 6.2% or higher.
The fifth modified fibroin may have a secretory signal for releasing a protein produced in a recombinant protein production system to the outside of a host. A sequence of the secretory signal can be suitably set according to the type of the host.
The sixth modified fibroin has an amino acid sequence in which a content of glutamine residues is reduced, compared to the naturally derived fibroin.
It is preferable that the sixth modified fibroin contains at least one motif selected from a GGX motif and a GPGXX motif in the amino acid sequence of REP.
In a case where the sixth modified fibroin contains the GPGXX motif in REP, a content rate of the GPGXX motifs is generally 1% or higher. The content rate of the GPGXX motif may be 5% or higher and is preferably 10% or higher. The upper limit of the content rate of the GPGXX motifs is not particularly limited, and may be 50% or lower or 30% or lower.
In the present specification, the “content rate of the GPGXX motifs” is a value calculated by the following method.
The content rate of the GPGXX motifs in fibroin having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif (modified fibroin or naturally derived fibroin) is calculated as s/t, in a case where a number which is three times a total number of the GPGXX motifs (that is, corresponding to the total number of G's and P's in the GPGXX motifs) contained in regions of all REP's contained in the domain sequence excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence is denoted by s, and a total number of amino acid residues in all REP's in the domain sequence excluding the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence and further excluding the (A)n motifs is denoted by t.
In the calculation of the content rate of the GPGXX motifs, “the domain sequence excluding the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence” is targeted, because “the sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence” (a sequence corresponding to REP) may contain a sequence having a low correlation with the sequence characteristic of fibroin, and in a case where m is small (that is, in a case where the domain sequence is short), the sequence may affect the calculation result of the content rate of the GPGXX motifs, and such effect needs to be eliminated. When a “GPGXX motif” is located at the C-terminus of REP, even in a case where “XX” is, for example, “AA”, the motif is regarded as a “GPGXX motif”.
The content rate of glutamine residues in the sixth modified fibroin is preferably 9% or lower, more preferably 7% or lower, even more preferably 4% or lower, and particularly preferably 0%.
In the present specification, the “content rate of glutamine residues” is a value calculated by the following method.
The content rate of glutamine residues in fibroin having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif (modified fibroin or naturally derived fibroin) is calculated as u/t, in a case where a total number of glutamine residues contained in regions of all REP's contained in the domain sequence excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence (a sequence corresponding to the “region A” in
The domain sequence of the sixth modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of glutamine residues in REP are deleted or substituted by other amino acid residues, compared to the naturally derived fibroin.
“Other amino acid residues” may be any amino acid residues other than glutamine residues, and an amino acid residue having a higher hydropathy index than the glutamine residue is preferred. The hydropathy indices of amino acid residues are as indicated in Table 1.
As indicated in Table 1, examples of the amino acid residue having a higher hydropathy index than the glutamine residue can include an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A) is more preferred, and an amino acid residue selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) is even more preferred.
In the sixth modified fibroin, hydrophobicity of REP is preferably −0.8 or higher, more preferably −0.7 or higher, even more preferably 0 or higher, still more preferably 0.3 or higher, and particularly preferably 0.4 or higher. The upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or lower or 0.7 or lower.
In the present specification, the “hydrophobicity of REP” is a value calculated by the following method.
The hydrophobicity of REP in fibroin having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A), motif (modified fibroin or naturally derived fibroin) is calculated as v/t, in a case where a sum of hydropathy indices of all amino acid residues in regions of all REP's contained in the domain sequence excluding a sequence from the (A)n motif located closest to the C-terminal side to the C-terminus of the domain sequence (a sequence corresponding to the “region A” in
In addition to the modification corresponding to a deletion of one or a plurality of glutamine residues in REP and/or a substitution of one or a plurality of glutamine residues in REP with other amino acid residues, compared to the naturally derived fibroin, further amino acid sequence modification may be performed on the domain sequence of the sixth modified fibroin, which corresponds to a substitution, a deletion, an insertion, and/or an addition of one or a plurality of amino acid residues.
The sixth modified fibroin can be obtained from, for example, a cloned gene sequence for the naturally derived fibroin by deleting one or a plurality of glutamine residues in REP and/or substituting one or a plurality of glutamine residues in REP with other amino acid residues. It is also possible to obtain the sixth modified fibroin by, for example, designing an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of glutamine residues in REP in the amino acid sequence of the naturally derived fibroin are deleted and/or one or a plurality of glutamine residues in REP in the amino acid sequence of the naturally derived fibroin are substituted by other amino acid residues and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
More specific examples of the sixth modified fibroin can include modified fibroin having (6-i) an amino acid sequence set forth in SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917), or SEQ ID NO: 42 (Met-PRT1028) or modified fibroin having (6-ii) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42.
The modified fibroin of (6-i) will be described. The amino acid sequence set forth in SEQ ID NO: 25 is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410) with VL's. The amino acid sequence set forth in SEQ ID NO: 26 is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 7 with TS's and substituting the remaining Q's with A's. The amino acid sequence set forth in SEQ ID NO: 27 is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 7 with VL's and substituting the remaining Q's with I's. The amino acid sequence set forth in SEQ ID NO: 28 is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 7 with VI's and substituting the remaining Q's with L's. The amino acid sequence set forth in SEQ ID NO: 29 is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 7 with VF's and substituting the remaining Q's with I's.
The amino acid sequence set forth in SEQ ID NO: 30 is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 8 (Met-PRT525) with VL's. The amino acid sequence set forth in SEQ ID NO: 31 is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 8 with VL's and substituting the remaining Q's with I's.
The amino acid sequence set forth in SEQ ID NO: 32 is obtained by substituting all QQ's with VF's in a sequence in which a region of 20 domain sequences existing in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410) is repeated twice and substituting the remaining Q's with I's.
The amino acid sequence set forth in SEQ ID NO: 41 (Met-PRT917) is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 7 with LI's and substituting the remaining Q's with V's. The amino acid sequence set forth in SEQ ID NO: 42 (Met-PRT1028) is obtained by substituting all QQ's in the amino acid sequence set forth in SEQ ID NO: 7 with IF's and substituting the remaining Q's with T's.
Content rates of glutamine residues in the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, and SEQ ID NO: 42 are all 9% or lower (Table 2).
The modified fibroin of (6-i) may consist of the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42.
The modified fibroin of (6-ii) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42. The modified fibroin of (6-ii) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif. It is preferable that the sequence identity is 95% or higher.
The content rate of glutamine residues in the modified fibroin of (6-ii) is preferably 9% or lower. Furthermore, the content rate of the GPGXX motifs in the modified fibroin of (6-ii) is preferably 10% or higher.
The sixth modified fibroin may have a tag sequence at one or both of the N-terminus and the C-terminus thereof. By having a tag sequence, isolation, immobilization, detection, visualization, and the like of the modified fibroin become possible.
More specific examples of the modified fibroin having a tag sequence can include modified fibroin having (6-iii) an amino acid sequence set forth in SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917), or SEQ ID NO: 44 (PRT1028) or modified fibroin having (6-iv) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44.
The amino acid sequences set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 are obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (which has a His-tag sequence and a hinge sequence) to the N-termini of the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, and SEQ ID NO: 42, respectively. Since only the tag sequence is added to the N-termini, the content rates of glutamine residues do not change, and the content rates of glutamine residues in the amino acid sequences set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 are all 9% or lower (Table 3).
The modified fibroin of (6-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44.
The modified fibroin of (6-iv) has an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44. The modified fibroin of (6-iv) is also a protein having a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif. It is preferable that the sequence identity is 95% or higher.
The content rate of glutamine residues in the modified fibroin of (6-iv) is preferably 9% or lower. Furthermore, the content rate of the GPGXX motifs in the modified fibroin of (6-iv) is preferably 10% or higher.
The sixth modified fibroin may have a secretory signal for releasing a protein produced in a recombinant protein production system to the outside of a host. A sequence of the secretory signal can be suitably set according to the type of the host.
The modified fibroin may have at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin.
The modified fibroin may be hydrophilic modified fibroin or hydrophobic modified fibroin. In the present specification, the “hydrophobic modified fibroin” is modified fibroin of which a value calculated by obtaining a sum of hydropathy indices (HI's) of all amino acid residues constituting the modified fibroin and then dividing the sum by a total number of amino acid residues (average HI) is larger than 0. The hydropathy indices are as indicated in Table 1. In addition, the “hydrophilic modified fibroin” is modified fibroin of which the average HI is 0 or lower. From the viewpoint of excellent burn resistance, the modified fibroin is preferably hydrophilic modified fibroin, and from the viewpoint of excellent hygroscopic heat-generating properties, the modified fibroin is preferably hydrophobic modified fibroin.
Examples of the hydrophobic modified fibroin can include modified fibroin having an amino acid sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43, or an amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 44.
Examples of the hydrophilic modified fibroin can include modified fibroin having an amino acid sequence set forth in SEQ ID NO: 4, an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, an amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, an amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, an amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or an amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
The artificial fibroin fiber according to the present embodiment may contain one kind of the modified fibroin alone or a combination of two or more kinds thereof.
(Method for Producing Modified Fibroin)
All of the modified fibroin according to the embodiment can be produced by expressing a nucleic acid encoding the modified fibroin using a host transformed with an expression vector having the nucleic acid sequence and one or a plurality of regulatory sequences operatively linked to the nucleic acid sequence.
A method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid can be produced by a method of performing amplification using a gene encoding natural fibroin by polymerase chain reaction (PCR) to clone the gene and modifying the gene by a genetic engineering procedure, or by a method of chemically synthesizing the nucleic acid. The method of chemically synthesizing the nucleic acid is not particularly limited, and for example, a gene can be chemically synthesized by a method of linking, by PCR or the like, oligonucleotides automatically synthesized with AKTA oligopilot plus 10/100 (GE Healthcare Japan Corporation) or the like based on information on the amino acid sequence of fibroin obtained from NCBI web database or the like. In this case, in order to allow easy purification and/or confirmation of the modified fibroin, a nucleic acid may be synthesized which encodes modified fibroin consisting of an amino acid sequence including the above amino acid sequence with an amino acid sequence consisting of a start codon and a His10-tag added to the N-terminus thereof.
The regulatory sequence is a sequence which controls the expression of the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding site, a transcription terminator sequence, and the like) and can be suitably selected according to the type of the host. As a promoter, an inducible promoter that can function in a host cell and induce the expression of the modified fibroin may be used. The inducible promoter is a promoter that can control transcription according to the presence of an inducer (expression inducing agent), absence of a repressor molecule, or a physical factor such as an increase or decrease in a temperature, osmotic pressure, or a pH value.
The type of the expression vector can be suitably selected according to the type of the host, and examples thereof can include a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. An expression vector which is capable of autonomously replicating in the host cell or integrating into the host chromosome and has a promoter at a position where the nucleic acid encoding the modified fibroin can be transcribed is suitably used.
As the host, any one of a prokaryote and a eukaryote such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
Preferable examples of the prokaryotic host can include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like. Examples of the microorganism belonging to the genus Escherichia can include Escherichia coli and the like. Examples of the microorganism belonging to the genus Brevibacillus can include Brevibacillus agri and the like. Examples of the microorganism belonging to the genus Serratia can include Serratia liquefaciens and the like. Examples of the microorganism belonging to the genus Bacillus can include Bacillus subtilis and the like. Examples of the microorganism belonging to the genus Microbacterium can include microbacterium ammoniaphilum and the like. Examples of the microorganism belonging to the genus Brevibacterium can include Brevibacterium divaricatum and the like. Examples of the microorganism belonging to the genus Corynebacterium can include Corynebacterium ammoniagenes and the like. Examples of the microorganism belonging to the genus Pseudomonas can include Pseudomonas putida and the like.
In a case where a prokaryote is used as the host, examples of the vector for introducing the nucleic acid encoding the modified fibroin can include pBTrp2 (manufactured by Boehringer Mannheim GmbH), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (JP 2002-238569 A), and the like.
Examples of the eukaryotic host can include yeast and filamentous fungi (mold or the like). Examples of the yeast can include yeasts belonging to the genera Saccharomyces, Pichia, Schizosaccharomyces, and the like. Examples of the filamentous fungi can include filamentous fungi belonging to the genera Aspergillus, Penicillium, Trichoderma, and the like.
In a case where a eukaryote is used as the host, examples of the vector for introducing the nucleic acid encoding the modified fibroin can include YEP13 (ATCC37115), YEp24 (ATCC37051), and the like. Any method can be used as a method for introducing the expression vector into the host cell, as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], an electroporation method, a spheroplast method, a protoplast method, a lithium acetate method, a competent method, and the like can be used.
As a method for expressing the nucleic acid by the host transformed with the expression vector, secretory production, fusion protein expression, or the like can be performed based on the method described in Molecular Cloning, 2nd edition, in addition to direct expression.
The modified fibroin can be produced by, for example, culturing the host transformed with the expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. A method for culturing the host in the culture medium can be performed according to a method generally used in culturing a host.
In a case where the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, any one of a natural medium and a synthetic medium may be used as the culture medium, as long as it is a medium containing a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host and capable of efficiently performing the culturing of the host.
Any carbon source that can be assimilated by the transformed microorganism may be used, and for example, carbohydrates such as glucose, fructose, sucrose, molasses containing glucose, fructose, and sucrose, starch, and a starch hydrolyzate, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used. As the nitrogen source, for example, ammonia, ammonium salts of an inorganic acid or organic acid, such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, a meat extract, a yeast extract, corn steep liquor, a casein hydrolyzate, soybean meal and a soybean meal hydrolyzate, and various fermentative bacteria cells and digests thereof can be used. As the inorganic salts, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, iron(II) sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.
The culture of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be performed under, for example, an aerobic condition such as shaking culture or deep aeration stirring culture. A culture temperature is, for example, 15° C. to 40° C. Culture time is generally 16 hours to 7 days. It is preferable that a pH of the culture medium is maintained at 3.0 to 9.0 during the culture. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like.
In addition, during the culture, an antibiotic such as ampicillin and tetracycline may be added to the culture medium as necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as the promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside may be added to the medium, and when culturing a microorganism transformed with an expression vector using a trp promoter, indoleacrylic acid may be added to the medium.
Isolation and purification of the expressed modified fibroin can be performed by a method that is generally used. For example, in a case where the modified fibroin is expressed in a state of being dissolved in the cells, the host cells are collected by centrifugation after the termination of the culture and suspended in an aqueous buffer. Then, the host cells are disrupted by an ultrasonic disintegrator, a French press, a Manton-Gaulin homogenizer, a Dyno-mill, or the like, and a cell-free extract is obtained. A method that is generally used in isolation and purification of proteins from a supernatant obtained by centrifugation of the cell-free extract, that is, a method such as a solvent extraction method, a salting-out method using ammonium sulfate, a desalination method, a precipitation method using an organic solvent, an anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei Corporation), a cation exchange chromatography method using a resin such as S-Sepharose FF (manufactured by Pharmacia), a hydrophobic chromatography method using a resin such as butyl-Sepharose and phenyl-Sepharose, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, and an electrophoresis method such as isoelectric focusing can be used alone or in combination to obtain a purified preparation.
Furthermore, in a case where the modified fibroin is expressed by forming an insoluble matter in the cells, the host cells are collected in the same manner, and then disrupted and subjected to centrifugation, thereby collecting the insoluble matter of the modified fibroin as a precipitated fraction. The insoluble matter of the modified fibroin thus collected can be solubilized by a protein denaturant. After the operation, a purified preparation of the modified fibroin can be obtained by the same isolation and purification methods as those described above. In a case where the modified fibroin is secreted outside the cells, the modified fibroin can be collected from a culture supernatant. That is, a culture supernatant is acquired by treating the culture by a method such as centrifugation, and a purified preparation can be obtained from the culture supernatant using the same isolation and purification methods as those described above.
(Artificial Fibroin Fiber)
The artificial fibroin fiber according to the present embodiment (hereinafter, may be referred to as an “uncrimped artificial fibroin fiber”) contains modified fibroin and is not crimped. The uncrimped artificial fibroin fiber is preferably an artificial spider silk fibroin fiber containing modified spider silk fibroin. The uncrimped artificial fibroin fiber is obtained by spinning the modified fibroin described above and contains the modified fibroin described above as a main component. The uncrimped artificial fibroin fiber according to the present embodiment is a fiber after the spinning and before the contact with the aqueous medium.
The uncrimped artificial fibroin fiber according to the present embodiment can be produced by a known spinning method. That is, for example, the modified fibroin produced according to the method described above is first added to a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and hexafluoroisopropanol (HFIP) along with an inorganic salt acting as a dissolution promoter and is dissolved therein, thereby preparing a dope solution. Next, the desired uncrimped artificial fibroin fiber can be obtained using the dope solution by performing spinning according to a known spinning method such as wet spinning, dry spinning, dry-wet spinning, and melt spinning. Preferable examples of a spinning method can include a wet spinning and dry-wet spinning.
A spinning method using the spinning apparatus 10 will be described. First, a dope solution 6 stored in a reservoir 7 is extruded from a spinneret 9 by a gear pump 8. On a laboratory scale, a cylinder may be filled with the dope solution, and the dope solution may be extruded from a nozzle using a syringe pump. Next, the extruded dope solution 6 is fed into a coagulation liquid 11 in a coagulation liquid tank 20 through an air gap 19, a solvent is removed, and the modified fibroin is coagulated, thus forming a fibrous coagulated body. The fibrous coagulated body is then fed into warm water 12 in a drawing bath 21 and drawn. A draw ratio is determined by a speed ratio between a feed nip roller 13 and a take-up nip roller 14. Thereafter, the drawn fibrous coagulated body is fed into the drying device 4 and dried in a thread guide 22, and the uncrimped artificial fibroin fiber is obtained as a yarn package 5. 18a to 18g are yarn guides.
Any solvent capable of desolvation may be used as the coagulation liquid 11, and examples thereof can include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone. The coagulation liquid 11 may suitably contain water. A temperature of the coagulation liquid 11 is preferably 0° C. to 30° C. In a case where a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the spinneret 9, an extrusion speed is preferably 0.2 to 6.0 ml/hour and more preferably 1.4 to 4.0 ml/hour per hole. A distance that the coagulated protein passes in the coagulation liquid 11 (substantially a distance from the yarn guide 18a to the yarn guide 18b) may be any length that allows desolvation to be efficiently performed, and is, for example, 200 to 500 mm. A take-up speed of the undrawn yarn may be, for example, 1 to 20 m/min and is preferably 1 to 3 m/min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes and is preferably 0.05 to 0.15 minutes. Furthermore, the drawing (pre-drawing) may be performed in the coagulation liquid 11. The coagulation liquid tank 20 may be provided in multiple stages, and the drawing may be performed in each stage or in a specific stage, as necessary.
As the drawing carried out when obtaining the uncrimped artificial fibroin fiber, for example, dry heat drawing is also adopted in addition to the pre-drawing performed in the coagulation liquid tank 20 and the wet heat drawing performed in the drawing bath 21 described above.
The wet heat drawing can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or during steam heating. A temperature may be, for example, 50° C. to 90° C. and preferably 75° C. to 85° C. In the wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times and preferably 2 to 8 times the original length.
The dry heat drawing can be performed using an electric tube furnace, a dry heat plate, or the like. A temperature may be, for example, 140° C. to 270° C. and preferably 160° C. to 230° C. In the dry heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times and preferably 1 to 4 times the original length.
The wet heat drawing and the dry heat drawing may each be performed independently, or may be performed in multiple stages or in combination. That is, the wet heat drawing and the dry heat drawing can be performed in a suitable combination such as a combination in which the first stage drawing is performed by the wet heat drawing and the second stage drawing is performed by the dry heat drawing, or a combination in which the first stage drawing is performed by the wet heat drawing, the second stage drawing is performed by the wet heat drawing, and further the third stage drawing is performed by the dry heat drawing.
A lower limit value of the final draw ratio is preferably any one of 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, and 9 times or more with respect to the undrawn yarn (or pre-drawn yarn), and the upper limit value 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 with respect to the undrawn yarn (or pre-drawn yarn). In a case where the uncrimped artificial fibroin fiber is a fiber spun at a draw ratio of 2 times or more, a shrinkage rate is further increased when the uncrimped artificial fibroin fiber is brought into contact with an aqueous medium, thereby being in a wet state.
(Raw Material Spun Yarn)
The raw material spun yarn according to the present embodiment includes the uncrimped artificial fibroin fiber. The raw material spun yarn may be a single yarn or a blended yarn such as a two-folded yarn. The type of the raw material spun yarn may be a spun yarn only consisting of the uncrimped artificial fibroin fibers (spun yarn of 100% modified fibroin), or may be a blended yarn of the uncrimped artificial fibroin fibers (fibers of 100% modified fibroin) and other fibers, for example, at least one selected from fibers consisting of crimped fibers such as wool or non-crimp fibers such as silk and synthetic fibers.
In a case where the raw material spun yarn only consists of the uncrimped artificial fibroin fibers, the raw material spun yarn can be obtained by a method including a cutting step of cutting the uncrimped artificial fibroin fibers (filaments) into an appropriate length to obtain modified fibroin staples and a spinning step of spinning the obtained modified fibroin staples.
The cutting step can be performed using any apparatus capable of cutting a modified fibroin fiber. Examples of such apparatus can include a desktop fiber cutting machine (s/NO. IT-160201-NP-300).
The length of the modified fibroin staple is not particularly limited, and is, for example, 20 mm or longer. The length of the modified fibroin staple may also be 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.
The spinning step can be performed by a known spinning method. Examples of the spinning method can include a cotton-type, worsted-type, or woollen-type method. Apparatuses used in these spinning methods are not particularly limited, and apparatuses that are generally used can be used. In addition, in the spinning step, the modified fibroin staples may first be subjected to opening or breaking by an opener or a breaker.
The spinning step can be carried out by, for example, performing carding on an assembly of the modified fibroin staples obtained in the cutting step (carding process) to prepare a sheet, preparing a sliver from the sheet, and then twisting the sliver to obtain a spun yarn (woollen-type method), or by preparing a sliver from the sheet and then aligning the sliver to obtain a spun yarn (worsted-type method).
In a case where the raw material spun yarn includes non-crimp fibers (such as silk) in addition to the uncrimped artificial fibroin fibers, the raw material spun yarn can be obtained by a method including a cutting step of cutting each of the uncrimped artificial fibroin fibers (filaments) and the additional non-crimp fibers into appropriate lengths to obtain modified fibroin staples and staples of the additional non-crimp fibers, respectively, and a spinning step of blending the obtained staples and performing spinning. The spinning may be performed after subjecting the staples of the additional non-crimp fibers to mechanical crimping or the like to obtain crimped fibers before the spinning. The spinning step is as described above.
In a case where the raw material spun yarn includes crimped fibers (such as wool) in addition to the uncrimped artificial fibroin fibers, it is preferable that the method for obtaining the raw material spun yarn includes a cutting step of cutting each of the uncrimped artificial fibroin fibers (filaments) and the crimped fibers into appropriate lengths to obtain modified fibroin staples and staples of the crimped fibers, respectively, and a step of blending the obtained staples and performing spinning by the woollen-type method. In this case, the uncrimped artificial fibroin fibers and the wool can be entangled by using the crimping in the crimped fibers such as wool.
In order to allow the uncrimped artificial fibroin fibers and additional fibers to be easily disentangled, an oil may adhere thereto in advance, before the spinning step. The oil adherence can be carried out in any stage in the production process. For example, the oil adherence can be carried out before the cutting step, simultaneously with the cutting step, or after the cutting step. The oil is not particularly limited, and any oil can be used as long as it is a known oil used for general purposes of imparting processability or functionality, such as purposes of preventing static charge, reducing friction, imparting softness, and imparting a water-repellent property.
[Step (b) ]
Step (b) is a step of crimping the uncrimped artificial fibroin fiber (hereinafter, may be referred to as the “artificial fibroin fiber”) by bringing the raw material spun yarn into contact with the aqueous medium (hereinafter, may be referred to as “water-crimping”). In addition to bringing the raw material spun yarn into contact with the aqueous medium without processing the raw material spun yarn, the water-crimping step also includes crimping raw material spun yarn by preparing an article such as various structural objects or molded products including a knitted fabric using the raw material spun yarn, and then bringing the article into contact with the aqueous medium.
The aqueous medium is a liquid or gas (steam) medium containing water (including water vapor). The aqueous medium may be water or a liquid mixture of water and a hydrophilic medium. Furthermore, as the hydrophilic medium, for example, a volatile solvent such as ethanol and methanol or a vapor thereof can be used. The aqueous medium may be a liquid mixture of water and a volatile solvent such as ethanol and methanol, and is preferably water or a liquid mixture of water and ethanol. By using an aqueous medium containing a volatile solvent or a vapor thereof, a drying speed after the water-crimping can be increased, and it is possible to impart a soft texture to the finally obtained protein spun yarn. A ratio between water and the volatile solvent or a vapor thereof is not particularly limited, and for example, a mass ratio of water:volatile solvent or vapor thereof may be 10:90 to 90:10. A proportion of water is preferably 30 mass % or higher and may be 40 mass % or 50 mass % or higher. In a case where the aqueous medium is a liquid, it is preferable that an oil is dispersed in the aqueous medium. In this case, the water-crimping and oil adhesion can be simultaneously performed. As the oil, any oil can be used as long as it is a known oil used for general purposes of imparting processability or functionality, such as purposes of preventing static charge, reducing friction, imparting softness, and imparting a water-repellent property. The amount of the oil is not particularly limited, and may be, for example, 1 to 10 mass % or 2 to 5 mass % with respect to the total amount of the oil and the aqueous medium.
The aqueous medium is preferably a liquid or a gas which is at a temperature of 10° C. to 230° C. and contains water (including water vapor). A temperature of the aqueous medium may be 10° C. or higher, 25° C. or higher, 40° C. or higher, 60° C. or higher, or 100° C. or higher, and may be 230° C. or lower, 120° C. or lower, or 100° C. or lower. More specifically, in a case where the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably 100° C. to 230° C. and more preferably 100° C. to 120° C. In a case where the steam of the aqueous medium is at a temperature of 230° C. or lower, thermal denaturation of a protein filament can be prevented. In a case where the aqueous medium is a liquid, the temperature of the aqueous medium is preferably 10° C. or higher, 25° C. or higher, or 40° C. or higher from the viewpoint of efficient crimping, and is preferably 60° C. or lower from the viewpoint of maintaining a fiber strength of the protein filament high.
A duration of the contact with the aqueous medium is not particularly limited and may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer. From the viewpoint of productivity, the duration is preferably 10 minutes or shorter. Furthermore, it is considered that, in the case of the steam, a higher shrinkage rate is obtained within a shorter period of time compared to the liquid. The contact with the aqueous medium may be performed under normal pressure or under reduced pressure (for example, in vacuum).
Examples of a method for contacting the aqueous medium can include a method of immersing the raw material spun yarn in the aqueous medium, a method of spraying the steam of the aqueous medium onto the raw material spun yarn, a method of exposing the raw material spun yarn to an atmosphere filled with the steam of the aqueous medium, and the like. In a case where the aqueous medium is a steam, the contact of the aqueous medium with the raw material spun yarn can be performed by using a general steam setting apparatus. Specific examples of the steam setting apparatus can include an apparatus such as product name: FMSA-type steam setter (manufactured by FUKUSHIN KOUGYO. Co., Ltd) and product name: EPS-400 (manufactured by Tsujii Senki Kogyo Co. Ltd.). Specific examples of a method for crimping the artificial fibroin fiber using the steam of the aqueous medium can include a method including storing the raw material spun yarn in a predetermined storage chamber and introducing the steam of the aqueous medium into the storage chamber, thus allowing the steam to contact the raw material spun yarn, while adjusting a temperature in the storage chamber to the predetermined temperature (for example, 100° C. to 230° C.).
Note that the step of crimping by the contact with the aqueous medium is performed in a state where no tensile force is applied to the raw material spun yarn (no tension is applied in the axial direction of the fiber) or in a state where only a predetermined amount of tensile force is applied to the raw material spun yarn (only a predetermined amount of tension is applied in the axial direction of the fiber). In this case, a degree of crimping can be controlled by adjusting the tensile force applied to the raw material spun yarn. Examples of a method for adjusting the tensile force applied to the raw material spun yarn can include a method of adjusting a load applied to the raw material spun yarn by suspending weights having various weights on the raw material spun yarn, a method of variously changing a degree of looseness of the raw material spun yarn while fixing both ends thereof in a loosened state, a method of appropriately changing a winding force (clamping force on a winding body such as a paper tube or a bobbin) of the raw material spun yarn while the raw material spun yarn is wound on the paper tube or the bobbin, and the like.
The crimping step may further include drying after the raw material spun yarn is brought into contact with the aqueous medium. A drying method is not particularly limited, and the drying may be natural drying or drying by hot wind or hot roller. A drying temperature is not particularly limited, and may be, for example, 20° C. to 150° C. The drying temperature is preferably 40° C. to 120° C. and more preferably 60° C. to 100° C.
(Shrinkage Rate of Artificial Fibroin Fiber)
By bringing the artificial fibroin fiber (fiber after the spinning and before the contact with the aqueous medium) into contact with the aqueous medium, the artificial fibroin fiber can be irreversibly crimped. Furthermore, the artificial fibroin fiber can be further crimped by being dried after the contact with the aqueous medium.
It is considered that the irreversible shrinkage of the artificial fibroin fiber (the “primary shrinkage” in
A dry shrinkage rate of the artificial fibroin fiber according to the present embodiment, which is defined by the following equation, may be higher than 7%.
Dry shrinkage rate={1−(length of artificial fibroin fiber brought into dry state after contact with aqueous medium/length of artificial fibroin fiber before contact with aqueous medium)}×100(%)
The dry shrinkage rate of the artificial fibroin fiber according to the present embodiment may be 8% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 37% or higher, 38% or higher, or 39% or higher. An upper limit of the dry shrinkage rate is not particularly limited and may be 80% or lower, 70% or lower, 60% or lower, 50% or lower, or 40% or lower.
A wet shrinkage rate of the artificial fibroin fiber according to the present embodiment, which is defined by the following equation, may be 2% or higher.
Wet shrinkage rate={1−(length of artificial fibroin fiber brought into wet state by contact with aqueous medium/length of artificial fibroin fiber after spinning and before contact with aqueous medium)}×100(%)
The wet shrinkage rate of the artificial fibroin fiber according to the present embodiment may be 2.5% or higher, 3% or higher, 3.5% or higher, 4% or higher, 4.5% or higher, 5% or higher, 5.5% or higher, or 6% or higher. An upper limit of the wet shrinkage rate is not particularly limited and may be 80% or lower, 60% or lower, 40% or lower, 20% or lower, 10% or lower, 7% or lower, 6% or lower, 5% or lower, 4% or lower, or 3% or lower.
In the production method according to the present invention, crimping is performed after the spinning step such as the carding process, and therefore, weakening of the crimping in the crimped artificial fibroin fibers, which is attributable to stretching, does not occur, and sufficient interlacing between fibers can be secured. Thus, protein spinning capable of securing a stable strength can be provided.
The protein spun yarn obtained by the production method according to the present invention exhibits a comparatively soft touch due to the water-crimping. In addition, since the artificial fibroin fibers have been brought into contact with moisture (aqueous medium), dimension change (shrinkage) of the spun yarn due to absorption of moisture during storage after the production of the spun yarn or during a process of manufacturing a product (such as a knitted fabric) using the spun yarn can be prevented.
Application of the protein spun yarn obtained by the production method according to the present invention is expected in clothing materials, medical hygiene products, interior products, bedding, ornaments, bags, accessories, general merchandise, vehicle parts, composite articles with other materials such as resin, and the like.
Hereinafter, the present invention will be more specifically described based on Examples. However, the present invention is not limited to the following Examples.
<Production Example of Artificial Spider Silk Protein (Artificial Spider Silk Fibroin) Filament>
(1) Preparation of Plasmid-Expressing Strain
Modified fibroin (hereinafter, also referred to as “PRT799”) having an amino acid sequence set forth in SEQ ID NO: 13 was designed based on the base sequence and the amino acid sequence of Nephila clavipes-derived fibroin (GenBank accession number: P46804.1, GI: 1174415). The amino acid sequence set forth in SEQ ID NO: 13 has an amino acid sequence obtained by performing a substitution, an insertion, and a deletion of amino acid residues on the amino acid sequence of the Nephila clavipes-derived fibroin for the purpose of improving productivity, and further includes the amino acid sequence set forth in SEQ ID NO: 5 (tag sequence and hinge sequence) added to the N-terminus thereof.
Next, a nucleic acid encoding PRT799 was synthesized. An NdeI site was added to the nucleic acid at the 5′-end, and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cut at NdeI and EcoRI by restriction enzyme treatment and then recombined with a protein expression vector pET-22b(+), thereby obtaining an expression vector.
(2) Expression of Protein
Escherichia coli BLR(DE3) was transformed with the pET22b(+) expression vector including the nucleic acid encoding a protein having the amino acid sequence set forth in SEQ ID NO: 13. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a medium for seed culture containing ampicillin (Table 4) so that OD600 reached 0.005. A seed culture solution was obtained by performing flask culture until OD600 reached 5 (about 15 hours), while keeping a temperature of the culture solution at 30° C.
The seed culture solution was added to a jar fermenter to which 500 mL of a production medium (Table 5) was added so that OD600 reached 0.05. Culture was performed while keeping a temperature of the culture solution at 37° C. and controlling a pH to be constant at 6.9. A concentration of dissolved oxygen in the culture solution was also maintained at 20% of the saturation concentration of dissolved oxygen.
Immediately after glucose in the production medium was completely consumed, a feed solution (455 g/l L glucose and 120 g/l L yeast extract) was added at a speed of 1 mL/min. Culture was performed while keeping a temperature of the culture solution at 37° C. and controlling a pH to be constant at 6.9. The concentration of dissolved oxygen in the culture solution was also maintained at 20% of the saturation concentration of dissolved oxygen, and the culture was performed for 20 hours. The expression of the modified fibroin was then induced by adding 1 M isopropyl-(-thiogalactopyranoside (IPTG) to the culture solution at a final concentration of 1 mM. When 20 hours have passed since the addition of IPTG, the bacterial cells were collected by centrifuging the culture solution. SDS-PAGE was performed using the bacterial cells prepared from the culture solutions obtained before the addition of IPTG and after the addition of IPTG, and the expression of the target modified fibroin which depended on the addition of IPTG was confirmed by appearance of a band of the size of the target modified fibroin.
(3) Purification of Protein
Bacterial cells that were collected two hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). After washing, the bacterial cells were suspended in a 20 mM Tris-HCl buffer solution (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The disrupted cells were centrifuged, thus obtaining a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer solution (pH 7.4) until the precipitate became highly pure. The washed precipitate was suspended in an 8 M guanidine buffer solution (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, and 1 mM Tris-HCl, pH 7.0) at a concentration of 100 mg/mL, and the precipitate was dissolved by stirring with a stirrer at 60° C. for 30 minutes. After the dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). A white aggregate protein obtained after the dialysis was collected by centrifugation, moisture was removed with a lyophilizer, and the lyophilized powder was collected, thereby obtaining the modified spider silk fibroin “PRT799”.
(4) Production of Protein Filament
The modified fibroin (PRT799) described above was added to DMSO at a concentration of 24 mass %, and then LiCl was added thereto as a dissolution promoter at a concentration of 4.0 mass %. Next, the modified fibroin was dissolved over 3 hours using a shaker to obtain a DMSO solution. A dope solution was obtained by removing dust and bubbles in the obtained DMSO solution. A solution viscosity of the dope solution was 5,000 cP (centipoise) at 90° C.
Known dry-wet spinning was performed using the dope solution obtained as described above and the spinning apparatus 10 shown in
Temperature of coagulation liquid (methanol): 5° C. to 10° C.
Draw ratio: 4.52 times
Drying temperature: 80° C.
A plurality of the artificial spider silk filaments wound onto the bobbin, which were obtained in the production example of the artificial spider silk protein, were bundled and cut to an average length of 50 mm using a desktop fiber cutting machine so as to prepare artificial spider silk protein staples. The artificial spider silk protein staples thus prepared were mixed in a manner that the orientations were disarranged while opening with a known opener, and then combed with an opening card until the artificial spider silk protein staples were formed into a single fibrous form (uniformly carded state). Next, the staples were fed into a four-protrusion woollen spinning carding machine, and in each of the movements from the first protrusion to the second protrusion, from the second protrusion to the third protrusion, and from the third protrusion to the fourth protrusion, the direction of the wave was changed by 90 degrees. The wave living the fourth protrusion was drawn and divided into tape shapes of sizes of 7 to 12 mm, and rubbed to be hardened into a sliver state in a condensed sliver state. Then, the sliver was subjected to drafting by a mule spinning machine and Z-twisted with a twist number of about 350 to obtain a spun yarn.
The uncrimped spun yarn was immersed in water at 40° C. for 1 minute so as to be curled for crimping, and then the crimped spun yarn was dried at 40° C. for 18 hours. As a result, a spun yarn with sufficient crimping was obtained.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-185695 | Sep 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/038434 | 9/27/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2020/067553 | 4/2/2020 | WO | A |
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| Number | Date | Country | |
|---|---|---|---|
| 20210388537 A1 | Dec 2021 | US |
