US20210040649A1

US20210040649A1 - Manufacturing Method for Protein Crimped Staple

Info

Publication number: US20210040649A1
Application number: US16/965,661
Authority: US
Inventors: Akito Sato; Yunosuke Abe
Original assignee: Spiber Inc
Current assignee: Spiber Inc
Priority date: 2018-01-31
Filing date: 2019-01-31
Publication date: 2021-02-11
Also published as: CN111699290A; WO2019151436A1; EP3748066A4; JPWO2019151436A1; EP3748066A1; JP7367977B2

Abstract

An object of the present invention is to provide a method for efficiently manufacturing a protein crimped staple from protein filaments at a low cost. A manufacturing method for a protein crimped staple according to the present invention includes: a) preparing an artificial fibroin filament containing a modified fibroin; b) cutting the artificial fibroin filament to obtain an artificial fibroin staple; and c) performing crimping by bringing the artificial fibroin filament into contact with an aqueous medium to crimp the artificial fibroin filament before the cutting or bringing the artificial fibroin staple into contact with an aqueous medium to crimp the artificial fibroin staple after the cutting.

Description

TECHNICAL FIELD

The present invention relates to a manufacturing method for a protein crimped staple and particularly relates to a manufacturing method for a crimped staple of an artificial fibroin including a modified fibroin.

BACKGROUND ART

A protein fiber, unlike a synthetic fiber, has biodegradability and low energy consumption for production and processing, and thus demands for various fields are expected to increase in response to the recent increase in environmental consciousness.
As the natural protein fiber, a filament such as silk and a staple such as wool are known. The former has a supple texture, and the latter has a soft feel and a heat retaining property, each of which has own characteristics thereof.
Recently, attempts have been made to process a protein fiber and apply the protein fiber to a wider range of applications. For example, in Patent Literatures 1 and 2, a method for manufacturing a long-fiber nonwoven fabric or a long-fiber crimped yarn by crimping the natural silk filament has been proposed. Further, the manufacturing of a protein crimped staple from protein filaments and obtaining spun yarn, nonwoven fabric, and the like using the protein crimped staple have also been studied in some fields.
As a method for obtaining a protein crimped staple from protein filaments, for example, a method of cutting a silk crimped filament crimped by the crimping processing method disclosed in the above patent literatures can be considered.

CITATION LIST

Patent Literature

[Patent Literature 1] Japanese Unexamined Patent Publication No. 2006-207069
[Patent Literature 2] Japanese Unexamined Patent Publication No. H9-119033

SUMMARY OF INVENTION

Technical Problem

However, since in the crimping method described in Patent Literature 1, a protein filament is crimped by a mechanical processing method such as an indentation method, in a case where a protein crimped staple is manufactured using this crimping method, there have been problems that a dedicated crimping device is required and the processing cost is high. Further, in the crimping method described in Patent Literature 2, it is necessary to perform a pretreatment for imparting a non-twisted latent crimping property to the natural silk prior to the crimping process. Therefore, in a case where this crimping method is used for manufacturing the protein crimped staple, there have been problems that the number of processes is increased and the productivity is inevitably decreased.
The present invention has been made in consideration of the above problems, and an object of the present invention is to provide a method for efficiently manufacturing a protein crimped staple from protein filaments at low cost.

Solution to Problem

The present invention relates to, for example, each of the following inventions.
[1] A manufacturing method for a protein crimped staple, including:
a) preparing an artificial fibroin filament containing a modified fibroin;
b) cutting the artificial fibroin filament to obtain an artificial fibroin staple; and
c) performing crimping by bringing the artificial fibroin filament into contact with an aqueous medium to crimp the artificial fibroin filament before the cutting or bringing the artificial fibroin staple into contact with an aqueous medium to crimp the artificial fibroin staple after the cutting.
[2] The manufacturing method for a protein crimped staple [1], in which a shrinkage rate after drying of the artificial fibroin filament defined by the following expression is more than 7%.
Shrinkage rate after drying={1−(length of artificial fibroin filament after being brought into contact with aqueous medium and subsequently dried/length of artificial fibroin filament before being brought into contact with aqueous medium)}×100 (%).
[3] The manufacturing method for a protein crimped staple according to [1] or [2], in which a shrinkage rate when wetted of the artificial fibroin filament defined by the following expression is 2% or more.
Shrinkage rate when wetted={1−(length of artificial fibroin filament when wetted by being brought into contact with aqueous medium/length of artificial fibroin filament before being brought into contact with aqueous medium)}×100 (%).
[4] The manufacturing method for a protein crimped staple according to any one of [1] to [3], in which the modified fibroin is a modified spider silk fibroin, and the artificial fibroin filament is an artificial spider silk fibroin filament.
[5] The manufacturing method for a protein crimped staple according to any one of [1] to [4], in which the aqueous medium used in the performing crimping is a liquid or gas containing water and having a temperature of 10° C. to 230° C.
[6] The manufacturing method for a protein crimped staple according to any one of [1] to [5], in which the performing crimping further includes performing drying after the bringing the artificial fibroin filament or the artificial fibroin staple into contact with the aqueous medium.
[7] The manufacturing method for a protein crimped staple according to any one of [1] to [6], in which the aqueous medium used in the performing crimping contains a volatile solvent.

Advantageous Effects of Invention

In the manufacturing method for a protein crimped staple of the present invention, by adopting a simple and unique crimping process of simply bring a raw material protein fiber into contact with an aqueous medium without using a dedicated crimping device, a protein crimped staple can be easily and efficiently manufactured at low cost.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram illustrating one example of a domain sequence of a modified fibroin.

FIG. 2 is a schematic diagram illustrating one example of a domain sequence of a modified fibroin.

FIG. 3 is a schematic diagram illustrating one example of a domain sequence of a modified fibroin.

FIG. 4 is an explanatory view schematically illustrating one example of a spinning device for manufacturing a protein filament.

FIG. 5 is a photograph of a protein crimped staple obtained in Example 1.

FIG. 6 is another photograph of a protein crimped staple obtained in Example 1.

FIG. 7 is a photograph of a protein uncrimped staple obtained in Comparative Example.

DESCRIPTION OF EMBODIMENTS

A manufacturing method for a protein crimped staple according to one aspect of the present invention includes a process a, a process b, and a process c, each of which will be described below, and the process b and the process c are in no particular order. That is, the method may be performed in an order of the process a, the process b, and the process c, or in an order of the process a, the process c, and the process b.
<Process a>
The process is a process of preparing an artificial fibroin filament containing a modified fibroin. Here, the filament (also referred to as “long fiber”) and the staple (also referred to as “short fiber”) are obvious to those skilled in the art.
(Modified Fibroin)
The modified fibroin according to the present embodiment is a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A)_nmotif—REP]_m—(A)_nmotif. In the modified fibroin, an amino acid sequence (an N-terminal sequence and a C-terminal sequence) may be further added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The
N-terminal sequence and the C-terminal sequence, although not limited thereto, are typically regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of amino acids of about 100 residues.
The term “modified fibroin” in the present specification means an artificially produced fibroin (an artificial fibroin). The modified fibroin may be a fibroin in which the domain sequence is different from the amino acid sequence of a fibroin derived from the natural fibroin or may be the same as the amino acid sequence of a fibroin derived from the natural fibroin. The “fibroin derived from the natural fibroin” referred to in the present specification is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A)_nmotif—REP]_m—(A)_nmotif
As the “modified fibroin”, an amino acid sequence of a fibroin derived from the natural fibroin may be directly used, a fibroin whose amino acid sequence has been modified based on an amino acid sequence of a fibroin derived from the natural fibroin (for example, a fibroin whose amino acid sequence has been modified by altering a cloned gene sequence of a fibroin derived from the natural fibroin) may be used, or a fibroin artificially designed and synthesized independently of a fibroin derived from the natural fibroin (for example, a fibroin having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence) may be used, as long as it has the amino acid sequence specified in the present embodiment.
The term “domain sequence” in the present specification refers to an amino acid sequence which produces a crystalline region (typically, corresponds to (A)_nmotif of an amino acid sequence) and an amorphous region (typically, corresponds to REP of an amino acid sequence) peculiar to fibroin and means an amino acid sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A)_nmotif—REP]_m—(A)_nmotif Here, the (A)_nmotif represents an amino acid sequence mainly including alanine residues, and the number of amino acid residues in the (A)_nmotif is 2 to 27. The number of amino acid residues in the (A)_nmotif 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. Further, the proportion of the number of alanine residues with respect to the total number of amino acid residues in the (A)_nmotif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the (A)_nmotif is composed of only alanine residues). In a plurality of (A)_nmotifs present in the domain sequence, at least seven of the (A)_nmotif may be composed of only alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. The REP may represent an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and may be an integer of 10 to 300. The plurality of (A)_nmotifs may have the same amino acid sequence or amino acid sequences different from each other. The plurality of REPs may have the same amino acid sequence or amino acid sequences different from each other.
The modified fibroin can be obtained, for example, by carrying out the modification of an amino acid sequence equivalent to the substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues with respect to, for example, a cloned gene sequence of a fibroin derived from the natural fibroin. The substitution, deletion, insertion, and/or addition of an amino acid residue may be carried out by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, the modifications may be carried out by methods described in literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).
The fibroin derived from the natural fibroin is a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A)_nmotif—REP]_m—(A)_nmotif, and specifically, for example, a fibroin produced by insects or spiders.
Examples of the fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Philosamia Cynthia ricini, Samia cynthia, Caligula japonica, Antheraea mylitta, and Antheraea assama; and hornet silk proteins discharged by larvae of Vespa simillima xanthoptera.
A more specific example of the fibroin produced by insects includes a silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), AAA27840.1 (amino acid sequence)).
Examples of the fibroin produced by spiders include spider silk proteins produced by spiders belonging to the genus Araneus such as i 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 minutes, spiders belonging to the genus Cyrtarachne such as Cyrtarachne bufo and Cyrtarachne inaequalis, spiders belonging to the genus Gasteracantha such as Gasteracantha kuhli 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 bruennich, 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 Cyrtophora 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 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 family Tetragnathidae such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include traction yarn proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
More specific examples of the spider silk protein produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank Accession No. AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence), U37520 (base sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank Accession No. ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank Accession No. AAL32472 (amino acid sequence), AF441245 (base sequence)), major ampullate spidroin 1 [derived from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ973155 (base sequence)) and major ampullate spidroin 2 [Euprosthenops australis] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence), minor ampullate silk protein 2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), and minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank Accession No. ABR37278.1 (amino acid sequence)).
As a further specified example of the fibroin derived from the natural fibroin, a fibroin whose sequence information is registered in NCBI GenBank may be mentioned. For example, sequences thereof may be confirmed by extracting sequences in which spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION among sequences containing INV as DIVISION in sequence information registered in NCBI GenBank, sequences in which a specific character string of a product is described from CDS, or sequences in which a specific character string is described from SOURCE to TISSUE TYPE.
The modified fibroin may be a modified silk fibroin (a modified silk protein obtained by modifying an amino acid sequence of a silk protein produced by silkworm), and a modified spider silk fibroin (a modified spider silk protein obtained by modifying an amino acid sequence of a spider silk protein produced by spiders). Among them, a modified spider silk fibroin is preferably used.
The specific examples of the modified fibroin include a modified fibroin derived from a large spinal canal thread protein produced in the major ampullate gland of a spider, a modified fibroin having a reduced content of the glycine residue, a modified fibroin having a reduced content of the (A)_nmotif, and a modified fibroin having a reduced content of the glycine residue and a reduced content of the (A)_nmotif
As the modified fibroin derived from a large spinal canal thread protein produced in the major ampullate gland of a spider, a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mis mentioned. In the modified fibroin derived from a large spinal canal thread protein produced in the major ampullate gland of a spider, the number of amino acid residues of the (A)_nmotif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, even more preferably an integer of 10 to 20, even further 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. In the modified fibroin derived from a large spinal canal thread protein produced in the major ampullate gland of a spider, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and still more preferably 20 to 100 residues, and even more preferably 20 to 75 residues. In the modified fibroin derived from a large spinal canal thread protein produced in the major ampullate gland of a spider, the number of residues in Formula 1: [(A)_nmotif—REP]_mis preferably 40% or more, more preferably 60% or more, and still more preferably 70% or more with respect to the total number of amino acid residues.
The modified fibroin derived from a large spinal canal thread protein produced in the major ampullate gland of a spider may be a polypeptide including an amino acid sequence unit represented by Formula 1: [(A)_nmotif—REP]_m, and being an amino acid sequence in which the C-terminal sequence has the amino acid sequence set forth in any of SEQ ID NOs: 14 to 16 or an amino acid sequence in which the C-terminal sequence has an amino acid sequence having 90% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 14 to 16.
The amino acid sequence set forth in SEQ ID NO: 14 is identical to the amino acid sequence consisting of 50 amino acid residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI). The amino acid sequence set forth in SEQ ID NO: 15 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminal of the amino acid sequence set forth in SEQ ID NO: 14. The amino acid sequence set forth in SEQ ID NO: 16 is identical to the amino acid sequence obtained by removing 29 residues from the C-terminal of the amino acid sequence set forth in SEQ ID NO: 14.
More specific examples of the modified fibroin derived from a large spinal canal thread protein produced in the major ampullate gland of a spider include a modified fibroin including (1-i) the amino acid sequence set forth in SEQ ID NO: 17 or (1-ii) the amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 17. The sequence identity is preferably 95% or more.
The amino acid sequence set forth in SEQ ID NO: 17 is an amino acid sequence obtained by approximately doubling repeating regions from the first repeating region to the 13th repeating region and performing mutation so that translation is terminated at the 1154th amino acid residue in an amino acid sequence obtained by adding the amino acid sequence (SEQ ID NO: 18) consisting of a start codon, a His10 tag, and a recognition site for HRV3C protease (human rhinovirus 3C protease) to the N-terminal of ADF3. The C-terminal amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 17 is identical to the amino acid sequence set forth in SEQ ID NO: 16.
The modified fibroin of (1-i) may consist of the amino acid sequence set forth in SEQ ID NO: 17.
The domain sequence of the modified fibroin having a reduced content of the glycine residue has an amino acid sequence with a reduced content of the glycine residue, as compared with a fibroin derived from the natural fibroin. It can be said that the modified fibroin has an amino acid sequence equivalent to an amino acid sequence in which at least one or a plurality of glycine residues in REP are substituted with other amino acid residues, as compared with a fibroin derived from the natural fibroin.
The domain sequence of the modified fibroin having a reduced content of the glycine residue may have an amino acid sequence equivalent to an amino acid sequence in which one glycine residue in at least one or the plurality of motif sequences, at least one of which is selected from GGX and GPGXX (where G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP, is substituted with other amino acid residue, as compared with a fibroin derived from the natural fibroin.
In the modified fibroin having a reduced content of the glycine residue, the proportion of the motif sequences in which the above-described glycine residue is substituted with other amino acid residue may be 10% or more with respect to the entire motif sequences.
The modified fibroin having a reduced content of the glycine residue may include a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mand have an amino acid sequence in which z/w is 30% or more, 40% or more, 50% or more, or 50.9% or more, in a case where the total number of amino acid residues consisting of XGX (where G represents a glycine residue and X represents an amino acid residue other than glycine) included in all REPs in the sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, is denoted by z, and the total number of amino acid residues in the amino acid sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, is denoted by w. The proportion of the number of alanine residues with respect to the total number of amino acid residues in the (A)_nmotif is 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even still more preferably 100% (which means that the (A)_nmotif consists of only alanine residues).
In the modified fibroin having a reduced content of the glycine residue, the content proportion of an amino acid sequence consisting of XGX is preferably increased by substituting one glycine residue in GGX motif with other amino acid residue. In the modified fibroin having a reduced content of the glycine residue, the content proportion of an amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, still more preferably 10% or less, even still more preferably 6% or less, still further preferably 4% or less, and particularly preferably 2% or less. The content proportion of an amino acid sequence consisting of GGX in a domain sequence can be calculated by the same method as the following method for calculating the content proportion (z/w) of the amino acid sequence consisting of XGX.
The calculation method of z/w will be described in more detail. First, in a fibroin (a modified fibroin or a fibroin derived from the natural fibroin) including a domain sequence represented by Formula 1: ([(A)_nmotif—REP]_m], the amino acid sequence consisting of XGX is extracted from all REPs included in a sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. The total number of amino acid residues constituting XGX is z. For example, in a case where 50 amino acid sequences consisting of XGX (without overlap) are extracted, z is 50×3=150. Further, for example, in a case where there exists an X (a central X) contained in two XGXs, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlapping portion (in the case of XGXGX, it is counted as 5 amino acid residues). w is the total number of amino acid residues included in the sequence excluding a sequence from the (A)_nmotif located closest to the C terminal side to the C terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence illustrated in FIG. 1, w is 4+50+4+100+4+10+4+20+4+30=230 (the (A)_nmotif located closest to the C-terminal side is excluded.). Next, z/w (%) can be calculated by dividing z by w.
In the modified fibroin having a reduced content of the glycine residue, z/w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, even still more preferably 70% or more, and still further preferably 80% or more. The upper limit of z/w is not particularly limited, but, for example, it may be 95% or less.
The modified fibroin having a reduced content of the glycine residue cab be obtained by, for example, modifying a cloned fibroin derived from the natural fibroin gene sequence so that at least a part of a base sequence encoding a glycine residue is substituted with other amino acid residue to encode other amino acid residue. In this case, one glycine residue in GGX motif and GPGXX motif may be selected as the glycine residue to be modified or may be substituted so that z/w is 50.9% or more. Alternatively, a modified fibroin may also be obtained, for example, by designing an amino acid sequence satisfying the above-described aspect based on the amino acid sequence of a fibroin derived from the natural fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, with respect to the amino acid sequence of a fibroin derived from the natural fibroin, in addition to the modification corresponding to the substitution of the glycine residue in REP with other amino acid residue, further modification of amino acid sequence corresponding to substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues may be carried out.
The other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than glycine residue, but it is preferably a hydrophobic amino acid residue such as valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine (M) residue, proline (P) residue, phenylalanine (F) residue, and tryptophan (W) residue, or a hydrophilic amino acid residues such glutamine (Q) residue, asparagine (N) residue, serine (S) residue, lysine (K) residue, and glutamic acid (E) residue, more preferably valine (V) residue, leucine (L) residue, isoleucine (I) residue, and glutamine (Q) residue, and still more preferably glutamine (Q) residue.
A more specific example of the modified fibroin having a reduced content of the glycine residues includes a modified fibroin including an amino acid sequence having (2-i) the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12.
The modified fibroin of (2-i) will be described. The amino acid sequence set forth in SEQ ID NO: 3 is obtained by substituting all GGXs in REP of the amino acid sequence set forth in SEQ ID NO: 1 equivalent to a fibroin derived from the natural fibroin with GQX. The amino acid sequence set forth in SEQ ID NO: 4 is obtained by deleting one of every two (A)_nmotifs from the N-terminal side to the C-terminal side in the amino acid sequence set forth in SEQ ID NO: 3 and further inserting one [(A)_nmotif—REP] just before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 10 is obtained by inserting two alanine residues at the C-terminal side of each (A)_nmotif of the amino acid sequence set forth in SEQ ID NO: 4, and further substituting a part of glutamine (Q) residues with serine (S) residues and deleting a part of amino acids on the N-terminal side so that the molecular weight thereof is approximately the same as that of SEQ ID NO: 4. The amino acid sequence set forth in SEQ ID NO: 12 is an amino acid sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating, four times, a region of 20 domain sequences (where several amino acid residues on the C-terminal side of the region are substituted) present in the amino acid sequence set forth in SEQ ID NO: 9.
The value of z/w in the amino acid sequence set forth SEQ ID NO: 1 (corresponds to a fibroin derived from the natural fibroin) is 46.8%. The values of z/w in the amino acid sequence set forth in SEQ ID NO: 3, the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 10, and the amino acid sequence set forth in SEQ ID NO: 12 are respectively 58.7%, 70.1%, 66.1%, and 70.0%. In addition, the values of x/y with a Giza ratio (described later) of 1:1.8 to 11.3 in the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 12 are respectively 15.0%, 15.0%, 93.4%, 92.7%, and 89.3%.
The modified fibroin of (2-i) may consist of the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12.
The modified fibroin of (2-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12. The modified fibroin of (2-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin of (2-ii) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, and in a case where the total number of amino acid residues in the amino acid sequence consisting of XGX (where G represents a glycine residue and X represents an amino acid residue other than glycine) included in REP is z, and the total number of amino acid residues in REP in the domain sequence is w, z/w is preferably 50.9% or more.
The above-described modified fibroin may include a tag sequence at either or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect, and visualize the modified fibroin.
The tag sequence may be, for example, an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. As a specific example of the affinity tag, a histidine tag (a His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are arranged and has a property of specifically binding to a metal ion such as nickel, and thus it can be used for isolation of a modified fibroin by a chelating metal chromatography. A specific example of the tag sequence may be an amino acid sequence set forth in SEQ ID NO: 5 (an amino acid sequence including a His tag).
In addition, a tag sequence such as glutathione-S-transferase (GST) that specifically binds to glutathione or a maltose binding protein (MBP) that specifically binds to maltose can also be used.
Further, an “epitope tag” utilizing an antigen-antibody reaction can also be used. By adding a peptide (an epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, a myc tag, and a FLAG tag. The modified fibroin can be easily purified with high specificity by utilizing an epitope tag.
It is also possible to use a tag sequence which can be cleaved with a specific protease. By treating a protein adsorbed via the tag sequence with a protease, it is also possible to recover a modified fibroin cleaved from the tag sequence.
A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (2-iii) the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
The amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13 are respectively amino acid sequences obtained by adding the amino acid sequence (including a His tag and a hinge sequence) set forth in SEQ ID NO: 5 to the N-terminal of the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 12.
The modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
The modified fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. The modified fibroin of (2-iv) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin of (2-iv) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, and in a case where the total number of amino acid residues in the amino acid sequence consisting of XGX (where G represents a glycine residue and X represents an amino acid residue other than glycine) included in REP is z, and the total number of amino acid residues in REP in the domain sequence is w, z/w is preferably 50.9% or more.
The above-mentioned modified fibroin may include a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The domain sequence of a modified fibroin having a reduced content of the (A)_nmotif has an amino acid sequence with a reduced content of the (A)_nmotif, as compared with a fibroin derived from the natural fibroin. It can be said that the domain sequence of the modified fibroin has an amino acid sequence equivalent to an amino acid sequence in which at least one or a plurality of (A)_nmotifs are deleted, as compared with a fibroin derived from the natural fibroin.
The modified fibroin having a reduced content of the (A)_nmotif may have an amino acid sequence equivalent to an amino acid sequence in which 10% to 40% of the (A)_nmotifs are deleted from a fibroin derived from the natural fibroin.
The domain sequence of the modified fibroin having a reduced content of the (A)_nmotif may have an amino acid sequence equivalent to an amino acid sequence obtained by deleting one of every one to three (A)_nmotifs from the N-terminal side to the C-terminal side, as compared with a fibroin derived from the natural fibroin.
The domain sequence of the modified fibroin having a reduced content of the (A)_nmotif may have an amino acid sequence equivalent to an amino acid sequence obtained by repeating deletion of at least two consecutive (A)_nmotifs and deletion of one (A)_nmotif in this order from the N-terminal side to the C-terminal side, as compared with a fibroin derived from the natural fibroin.
The domain sequence of the modified fibroin having a reduced content of the (A)_nmotif may have an amino acid sequence equivalent to an amino acid sequence obtained by deleting one of every two (A)_nmotifs from the N-terminal side to the C-terminal side.
The modified fibroin having a reduced content of the (A)_nmotif may include a domain sequence represented by Formula 1: [(A)_nmotif—REP]_m, and in a case where the number of amino acid residues of two [(A)_nmotif—REP] units adjacent to each other is sequentially compared from the N-terminal side to the C-terminal side and then the number of amino acid residues of one REP having a small number of amino acid residues is set to 1, the maximum total value of the number of amino acid residues of two [(A)_nmotif—REP] units adjacent to each other, in which the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3, is denoted by x, and the total number of amino acid residues in the domain sequence is denoted by y, the modified fibroin may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more. The proportion of the number of alanine residues with respect to the total number of amino acid residues in the (A)_nmotif is 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even still more preferably 100% (which means that the (A)_nmotif consists of only alanine residues).
The method for calculating x/y will be described in more detail with reference to FIG. 1. FIG. 1 illustrates a domain sequence obtained by removing an N-terminal sequence and a C-terminal sequence from a modified fibroin. The domain sequence has a sequence of, from the N-terminal side (left side), (A)_nmotif—first REP (50 amino acid residues)—(A)_nmotif—second REP (100 amino acid residues)—(A)_nmotif—third REP (10 amino acid residues)—(A)_nmotif—fourth REP (20 amino acid residues)—(A)_nmotif—fifth REP (30 amino acid residues)—(A)_nmotif sequence.
Two [(A)_nmotif—REP] units adjacent to each other are sequentially selected from the N-terminal side toward the C-terminal side so that the units are not overlapped with each other. In this case, an unselected [(A)_nmotif—REP] unit may be present. In FIG. 1, pattern 1 (comparison of first REP and second REP, and comparison of third REP and fourth REP), pattern 2 (comparison of first REP and second REP, and comparison of fourth REP and fifth REP), pattern 3 (comparison of second REP and third REP, and comparison of fourth REP and fifth REP), and pattern 4 (comparison of first REP and second REP). There are other selection methods other than these methods.
Subsequently, for each pattern, the number of amino acid residues of each REP in two selected [(A)_nmotif—REP] units adjacent to each other is compared. The comparison is performed by determining the ratio of the number of amino acid residues of one REP to the number of amino acid residues of the other REP having the smaller number of amino acid residues so that the number of amino acid residues in the other REP is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the second REP is 100/50=2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the fifth REP is 30/20=1.5.
In FIG. 1, in a case where one group of [(A)_nmotif—REP] units having the smaller number of amino acid residues is set to 1, the other group in which the ratio of the number of amino acid residues is 1.8 to 11.3 is indicated by a solid line. Hereinafter, this ratio is referred to as a Giza ratio. In a case where one group of [(A)_nmotif—REP] units having the smaller number of amino acid residues is set to 1, the other group in which the ratio of the number of amino acid residues is less than 1.8 or more than 11.3 is indicated by a broken line.
In each pattern, the total numbers of amino acid residues of two [(A)_nmotif—REP] units adjacent to each other indicated by solid lines are added (not only the number of REPs but also the number of the amino acid residues in the (A)_nmotif are added.) Then, the added total values are compared, and the total value (maximum value of the total values) of the pattern having the maximum total value is denoted by x. In the example illustrated in FIG. 1, the total value of the pattern 1 is the maximum.
Next, x/y (%) can be calculated by dividing x by y which is the total number of the amino acid residues of the domain sequence.
In the modified fibroin having a reduced content of the (A).
motif, x/y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, even still more preferably 70% or more, still further preferably 75% or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly limited, but for example, it may be 100% or less. In a case where the Giza ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more. In a case where the Giza ratio is 1:1.8 to 3.4, x/y is more preferably 77.1% or more. In a case where the Giza ratio is 1:1.9 to 8.4, x/y is still more preferably 75.9% or more. In a case where the Giza ratio is 1:1.9 to 4.1, x/y is even still more preferably 64.2% or more.
In a case where the modified fibroin having a reduced content of the (A)_nmotif is a modified fibroin in which at least seven (A)_nmotifs present in the domain sequence are composed of only alanine residues, x/y is preferably 46.4% or more, more preferably 50% or more, still more preferably 55% or more, even still more preferably 60% or more, still further preferably 70% or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly limited as long as it is 100% or less.
The modified fibroin having a reduced content of the (A)_nmotif can be obtained, for example, by deleting one or a plurality sequences encoding (A)_nmotif from a cloned gene sequence of a fibroin derived from the natural fibroin so that x/y is 64.2% or more. Alternatively, the modified fibroin having a reduced content of the (A)_nmotif may also be obtained, for example, by designing an amino acid sequence equivalent to an amino acid sequence obtained by deleting one or a plurality (A)_nmotifs so that x/y is 64.2% or more based on the amino acid sequence of a fibroin derived from the natural fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, with respect to the amino acid sequence of a fibroin derived from the natural fibroin, in addition to the modification corresponding to the deletion of the (A)_nmotif, further modification of amino acid sequence equivalent to substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues may be carried out.
A more specific example of the modified fibroin having a reduced content of the (A)_nmotif includes a modified fibroin including an amino acid sequence having (3-i) the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12.
The modified fibroin of (3-i) will be described. The amino acid sequence set forth in SEQ ID NO: 2 is obtained by deleting one of every two(A)_nmotifs from the N-terminal side to the C-terminal side in the amino acid sequence set forth in SEQ ID NO: 1 equivalent to a fibroin derived from the natural fibroin and by further inserting one [(A)_nmotif—REP] just before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 4 is obtained by substituting all GGXs in REP of the amino acid sequence set forth in SEQ ID NO: 2 with GQX. The amino acid sequence set forth in SEQ ID NO: 10 is obtained by inserting two alanine residues at the C-terminal side of each (A)_nmotif of the amino acid sequence set forth in SEQ ID NO: 4, and further substituting a part of glutamine (Q) residues with serine (S) residues and deleting a part of amino acids on the N-terminal side so that the molecular weight thereof is approximately the same as that of SEQ ID NO: 4. The amino acid sequence set forth in SEQ ID NO: 12 is an amino acid sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating, four times, a region of 20 domain sequences (where several amino acid residues on the C-terminal side of the region are substituted) present in the amino acid sequence set forth in SEQ ID NO: 9.
The value of x/y with a Giza ratio of 1:1.8 to 11.3 in the amino acid sequence set forth in SEQ ID NO: 1 (equivalent to a fibroin derived from the natural fibroin) is 15.0%. Both the values of x/y in the amino acid sequence set forth in SEQ ID NO: 2 and the value of x/y in the amino acid sequence set forth in SEQ ID NO: 4 are 93.4%. The value of x/y in the amino acid sequence set forth in SEQ ID NO: 10 is 92.7%. The value of x/y in the amino acid sequence set forth in SEQ ID NO: 12 is 89.3%. The values of z/w in the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 12 are respectively 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%.
The modified fibroin of (3-i) may consist of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12.
The modified fibroin of (3-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12. The modified fibroin of (3-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin of (3-ii) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, and in a case where the number of amino acid residues of two [(A)_nmotif—REP] units adjacent to each other is sequentially compared from the N-terminal side to the C-terminal side, then the number of amino acid residues of one REP having a small number of amino acid residues is set to 1, and the maximum total value of the added numbers of amino acid residues of two [(A)_nmotif—REP] units adjacent to each other, in which the ratio (1:1.8 to 11.3 as a Giza ratio) of the number of amino acid residues of the other REP is 1.8 to 11.3, 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 more.
The modified fibroin described above may include a tag sequence described above at either or both of the N-terminal and C-terminal
A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (3-iii) the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
The amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13 are respectively amino acid sequences obtained by adding the amino acid sequence (including a His tag) set forth in SEQ ID NO: 5 to the N-terminal of the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 12.
The modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
The modified fibroin of (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. The modified fibroin of (3-iv) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin of (3-iv) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, and in a case where the number of amino acid residues of two [(A)_nmotif—REP] units adjacent to each other is sequentially compared from the N-terminal side to the C-terminal side, then the number of amino acid residues of one REP having a small number of amino acid residues is set to 1, the maximum total value of the added numbers of amino acid residues of two [(A)_nmotif—REP] units adjacent to each other, in which the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3, 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 more.
The above-mentioned modified fibroin may include a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The domain sequence of the modified fibroin having a reduced content of the glycine residue and (A)_nmotif has an amino acid sequence having not only a reduced content of the (A)_nmotif but also having a reduced content of the glycine residue, as compared with a fibroin derived from the natural fibroin. It can be said that the domain sequence of the modified fibroin has an amino acid sequence equivalent to an amino acid sequence in which at least one or a plurality of (A)_nmotifs are deleted and further at least one or a plurality of glycine residues in REP are substituted with other amino acid residues, as compared with a fibroin derived from the natural fibroin. That is, the modified fibroin is a modified fibroin having both of the characteristics of a modified fibroin having a reduced content of the glycine residue and the characteristics of a modified fibroin having a reduced content of the (A)_nmotif Specific aspects and the like are as described for a modified fibroin having a reduced content of the glycine residue and a modified fibroin having a reduced content of the (A)_nmotif
A more specific example of the modified fibroin having a reduced content of the glycine residue and (A)_nmotif includes a modified fibroin including an amino acid sequence having (4-i) the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, or (4-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12. Specific aspects of the modified fibroin including the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12 are as described above.
A domain sequence of a modified fibroin according to another embodiment may have an amino acid sequence locally containing a region with a high hydropathy index equivalent to an amino acid sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues with a high hydropathy index and/or one or a plurality of amino acid residues with a high hydropathy index are inserted into REP, as compared with a fibroin derived from the natural fibroin.
It is preferable that the region locally having high hydropathy index is composed of two to four consecutive amino acid residues.
It is more preferable that the amino acid residues with a high hydropathy index are selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A).
The modified fibroin according to the present embodiment may further include an amino acid sequence equivalent to an amino acid sequence in which one or a plurality of amino acid residues are substituted, deleted, inserted and/or added, as compared with a fibroin derived from the natural fibroin, in addition to the modification of the amino acid sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues with a high hydropathy index and/or one or a plurality of amino acid residues with a high hydropathy index are inserted into REP, as compared with a fibroin derived from the natural fibroin of the present embodiment.
The modified fibroin according to the present embodiment may be obtained by, with respect to a cloned gene sequence of a fibroin derived from the natural fibroin, substituting one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydropathy index) with a hydrophobic amino acid residue (for example, amino acid residues having a positive hydropathy index), and/or inserting one or a plurality of hydrophobic amino acid residues into REP. Further, for example, the modified fibroin may also be obtained by, for example, designing an amino acid sequence equivalent to an amino acid sequence in which with respect to the amino acid sequence of a fibroin derived from the natural fibroin, one or a plurality of hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues and/or one or a plurality of hydrophobic amino acid residues are inserted into REP, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, with respect to the amino acid sequence a fibroin derived from the natural fibroin, in addition to the modification corresponding to the substitution of one or a plurality of hydrophilic amino acid residues in REP with hydrophobic amino acid residues and/or insertion of one or a plurality of hydrophobic amino acid residues into REP, further modification of amino acid sequence equivalent to substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues may be carried out.
Further, a modified fibroin according to another embodiment may include a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mand have an amino acid sequence in which p/q is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from an (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of the four consecutive amino acid residues is 2.6 or more is denoted by p, and the total number of amino acid residues contained in the sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q.
Regarding the hydropathy index of amino acid residues, known indices from (Hydropathy index: Kyte J, & Doolittle R (1982)“A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. 105-132) may be used as a reference. Specifically, the hydropathy index (hereinafter, also referred to as “HI”) of each amino acid is as shown in Table 1 below.

	TABLE 1

	Amino acid	HI

	Isoleucine (Ile)	4.5
	Valine (Val)	4.2
	Leucine (Leu)	3.8
	Phenylalanine (Phe)	2.8
	Cysteine (Cys)	2.5
	Methionine (Met)	1.9
	Alanine (Ala)	1.8
	Glycine (Gly)	−0.4
	Threonine (Thr)	−0.7
	Serine (Ser)	−0.8
	Tryptophan (Trp)	−0.9
	Tyrosine (Tyr)	−1.3
	Proline (Pro)	−1.6
	Histidine (His)	−3.2
	Asparagine (Asn)	−3.5
	Aspartic acid (Asp)	−3.5
	Glutamine (Gln)	−3.5
	Glutamic acid (Glu)	−3.5
	Lysine (Lys)	−3.9
	Arginine (Arg)	−4.5

The calculation method of p/q will be described in more detail. In the calculation, the sequence (hereinafter, also referred to as “sequence A”) excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by Formula 1: [(A)_nmotif—REP]. is used. First, in all REPs included in the sequence A, average values of hydropathy indices of the four consecutive amino acid residues are calculated. The average value of the hydropathy indices is obtained by dividing the total sum of HI of each of the amino acid residues contained in the four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydropathy indices is obtained for all of the four consecutive amino acid residues (each of the amino acid residues is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydropathy indices of the four consecutive amino acid residues is 2.6 or more is specified. Even in a case where a plurality of certain amino acid residues correspond to the “four consecutive amino acid residues having an average value of the hydropathy indices of 2.6 or more”, the amino acid residue is counted as one amino acid residue in the region. The total number of amino acid residues included in the region is denoted by p. The total number of amino acid residues included in the sequence A is denoted by q.
For example, in a case where the “four consecutive amino acid residues whose average value of the hydropathy indices is 2.6 or more” are extracted from 20 places (without overlap), in the region where the average value of the hydropathy indices of the four consecutive amino acid residues is 2.6 or more, the number of the four consecutive amino acid residues (without overlap) is 20, and thus p is 20×4=80. In addition, for example, in a case where two of the “four consecutive amino acid residues having an average value of the hydropathy indices of 2.6 or more” overlap by only one amino acid residue, in the region where the average value of the hydropathy indices of the four consecutive amino acid residues is 2.6 or more, the number of amino acid residues being included is 7 (p=2×4−1=7. “−1” corresponds to the subtraction of the overlapping portion). For example, in the case of the domain sequence shown in FIG. 2, since the number of the “four consecutive amino acid residues having an average value of the hydropathy indices of 2.6 or more”, which do not overlap, is 7, p is 7×4=28. Further, for example, in the case of the domain sequence illustrated in FIG. 2, q is 4+50+4+40+4+10+4+20+4+30=170 (the (A)_nmotif present closest to the C-terminal side can not be included). Next, p/q (%) can be calculated by dividing p by q. In the case of FIG. 2, p/q (%) is 28/170=16.47%.
In the modified fibroin according to the present embodiment, p/q is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, even still more preferably 20% or more, and still further preferably 30% or more. The upper limit of p/q is not particularly limited, but for example, it may be 45% or less.
The modified fibroin according to the present embodiment may be obtained by, for example, modifying an amino acid sequence of a cloned a fibroin derived from the natural fibroin to an amino acid sequence locally containing a region locally having a high hydropathy index by substituting one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydropathy index) with hydrophobic amino acid residues (for example, amino acid residues having a positive hydropathy index), and/or inserting one or a plurality of hydrophobic amino acid residues into REP, so that the p/q condition is satisfied. Alternatively, the modified fibroin may also be obtained, for example, by designing an amino acid sequence satisfying the p/q condition based on the amino acid sequence of a fibroin derived from the natural fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the substitution of one or a plurality of amino acid residues in REP with amino acid residues with a high hydropathy index and/or insertion of one or a plurality of amino acid residues with a high hydropathy index into REP, as compared with the amino acid sequence of a fibroin derived from the natural fibroin, further modification corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may be carried out.
The amino acid residue with a high hydropathy index 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), but is not particularly limited thereto.
Another specific example of the modified fibroin may be a modified fibroin including (5-iii) the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23, or (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.
The modified fibroin of (5-i) will be described. The amino acid sequence set forth in SEQ ID NO: 19 is obtained by deleting a part of the amino acid sequence of the consecutive alanine residues in the (A)_nmotif of a fibroin derived from the natural fibroin so that the number of the consecutive alanine residues in the (A)_nmotif is five. The amino acid sequence set forth in SEQ ID NO: 20 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 19, and deleting a part of the amino acids on the C-terminal side therefrom so that the molecular weight thereof is approximately the same as that of the amino acid sequence set forth in SEQ ID NO: 19. The amino acid sequence set forth in SEQ ID NO: 21 is obtained by inserting two alanine residues at the C-terminal side of each (A)_nmotif with respect to the amino acid sequence set forth in SEQ ID NO: 19, and further 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 approximately the same as that of the amino acid sequence set forth in SEQ ID NO: 19. The amino acid sequence set forth in SEQ ID NO: 22 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at one site for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 21. The amino acid sequence set forth in SEQ ID NO: 23 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 21.
The modified fibroin of (5-i) may consist of the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.
The modified fibroin of (5-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23. The modified fibroin of (5-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin of (5-ii) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23, and preferably has an amino acid sequence in which p/q is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of the four consecutive amino acid residues is 2.6 or more is denoted by p, and the total number of amino acid residues contained in the sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q.
The above-described modified fibroin may include a tag sequence at either or both of the N-terminal and C-terminal
A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (5-iii) the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or (5-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.
The amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 are respectively amino acid sequences obtained by adding the amino acid sequence (including a His tag and a hinge sequence) set forth in SEQ ID NO: 5 to the N-terminal of the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 23.
The modified fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.
The modified fibroin of (5-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. The modified fibroin of (5-iv) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_m. The sequence identity is preferably 95% or more.
The modified fibroin of (5-iv) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, and preferably has an amino acid sequence in which p/q is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of the four consecutive amino acid residues is 2.6 or more is denoted by p, and the total number of amino acid residues contained in the sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q.
The above-mentioned modified fibroin may include a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
A modified fibroin according to another embodiment has an amino acid sequence with a reduced content of the glutamine residue, as compared with a fibroin derived from the natural fibroin.
It is preferable that the modified fibroin according to the present embodiment includes at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
In a case where the modified fibroin according to the present embodiment includes a GPGXX motif in REP, a GPGXX motif content rate is usually 1% or more, may be 5% or more, and is preferably 10% or more. The upper limit of the GPGXX motif content rate is not particularly limited, may be 50% or less, and may be 30% or less.
In the present specification, the “GPGXX motif content rate” is a value calculated by the following method.
In a fibroin (a fibroin derived from the natural fibroin) including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A)_nmotif—REP]_m—(A)_nmotif, in a case where the number obtained by tripling the total number of the GPGXX motifs included in all REPs included in a sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence (that is, equivalent to the total number of G and P in the GPGXX motifs) is denoted by s, and the total number of amino acid residues in all REPs excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence and further excluding (A)_nmotifs is denoted by t, the GPGXX motif content rate is calculated as s/t.
For the calculation of the GPGXX motif content rate, the “sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence” is used to exclude the effect occurring due to the fact that the “sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal from the domain sequence” (sequence equivalent to REP) may include a sequence that is not correlated with the sequence characteristics of fibroin, which influences the calculation result of the GPGXX motif content rate in a case where m is small (that is, in case a where the domain sequence is short). In a case where a “GPGXX motif” is located at the C-terminal of REP, it is treated as “GPGXX motif” even in a case where “XX” is, for example, “AA”.
FIG. 3 is a schematic diagram showing a domain sequence of a modified fibroin. The calculation method of the GPGXX motif content rate will be specifically described with reference to FIG. 3. First, in a domain sequence of a modified fibroin (which is an [(A)_nmotif—REP]_m—(A)_nmotif] type) illustrated in FIG. 3, since all REPs are included in the “sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence” (in FIG. 3, shown as “region A”), the number of GPGXX motifs for calculating s is 7, and s is 7×3=21. Similarly, since all REPs are included in the “sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence” (in FIG. 1, shown as “region A”), t which is the total number of amino acid residues in all REPs excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence and further excluding (A)_nmotifs, is 50+40+10+20+30=150. Next, s/t (%) can be calculated by dividing s by t and is 21/150=14.0% in the case of the modified fibroin of FIG. 3.
In the modified fibroin according to the present embodiment, a glutamine residue content rate is preferably 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%.
In the present specification, the “glutamine residue content rate” is a value calculated by the following method.
In a fibroin (a modified fibroin or a fibroin derived from the natural fibroin) including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A)_nmotif—REP]_m—(A)_nmotif, in a case where the total number of glutamine residues included in all REPs included in a sequence (sequence equivalent to “region A” in FIG. 3) excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by u, and the total number of amino acid residues in all REPs excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence and further excluding (A)_nmotifs is denoted by t, the glutamine residue content rate is calculated as u/t. For the calculation of the glutamine residue content rate, the “sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence” is used for the same reason described above.
The domain sequence of the modified fibroin according to the present embodiment may include an amino acid sequence equivalent to an amino acid sequence in which one or a plurality of glutamine residues in REP are deleted or substituted with other amino acid residues, as compared with a fibroin derived from the natural fibroin.
The “other amino acid residue” may be an amino acid residue other than a glutamine residue but is preferably an amino acid residue having a higher hydropathy index than that of a glutamine residue. The hydropathy indices of amino acid residues are as shown in Table 1.
As shown in Table 1, amino acid residues having a higher hydropathy index than a glutamine residue 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 preferable, and an amino acid residue selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) is still more preferable.
In the modified fibroin according to the present embodiment, the hydrophobicity of REP is preferably −0.8 or more, more preferably −0.7 or more, still more preferably 0 or more, even still more preferably 0.3 or more, and particularly preferably 0.4 or more. The upper limit of the hydrophobicity of REP is not particularly limited, may be 1.0 or less, and may be 0.7 or less.
In the present specification, the “hydrophobicity of REP” is a value calculated by the following method.
In a fibroin (a modified fibroin or a fibroin derived from the natural fibroin) including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A)_nmotif—REP]_m—(A)_nmotif, in a case where the total sum of the hydropathy index of each amino acid residue included in all REPs included in a sequence (sequence equivalent to “region A” in FIG. 1) excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by v, and the total number of amino acid residues in all REPs excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence and further excluding (A)_nmotifs is denoted by t, the hydrophobicity is calculated as v/t. For the calculation of the hydrophobicity of REP, the “sequence excluding a sequence from the (A)_nmotif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence” is used for the same reason described above.
The domain sequence of the modified fibroin according to the present embodiment may further include an amino acid sequence equivalent to an amino acid sequence in which one or a plurality of amino acid residues are substituted, deleted, inserted and/or added, in addition to the modification of the amino acid sequence in which one or a plurality of glutamine residues in REP are deleted and/or one or a plurality of glutamine residues in REP are substituted with other amino acid residues, as compared with a fibroin derived from the natural fibroin.
The modified fibroin according to the present embodiment can be obtained by, for example, with respect to a cloned gene sequence of a fibroin derived from the natural fibroin, deleting one or a plurality of glutamine residues in REP and/or by substituting one or a plurality of glutamine residues in REP with other amino acid residues. Further, for example, the modified fibroin may also be obtained by designing an amino acid sequence equivalent to an amino acid sequence in which with respect to the amino acid sequence of a fibroin derived from the natural fibroin, one or a plurality of glutamine residues in REP are deleted and/or one or a plurality of glutamine residues in REP are substituted with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
A more specific example of the modified fibroin according to the embodiment of the present invention may be a modified fibroin including (6-i) the 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: 38, or SEQ ID NO: 39, or (6-ii) an amino acid sequence having 90% or more sequence identity with the 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: 38, or SEQ ID NO: 39.
The modified fibroin of (6-i) will be described.
The amino acid sequence (Met-PRT410) set forth in SEQ ID NO: 4 is a modified amino acid sequence obtained by changing the number of the consecutive alanine residues in the (A)_nmotif to five, or the like, so as to improve productivity, based on the base sequence and amino acid sequence of Nephila clavipes (GenBank Accession No.: P46804.1, GI: 1174415) which is a fibroin derived from the natural fibroin. However, since Met-PRT410 has no modification of glutamine residue (Q), the glutamine residue content rate thereof is the same as the glutamine residue content of a fibroin derived from the natural fibroin.
The amino acid sequence (M_PRT888) set forth in SEQ ID NO: 27 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VLs.
The amino acid sequence (M_PRT965) set forth in SEQ ID NO: 28 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with TSs and substituting the remaining Qs with As.
The amino acid sequence (M_PRT889) set forth in SEQ ID NO: 29 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VLs and substituting the remaining Qs with Is.
The amino acid sequence (M_PRT916) set forth in SEQ ID NO: 30 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VIs and substituting the remaining Qs with Ls.
The amino acid sequence (M_PRT918) set forth in SEQ ID NO: 31 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VFs and substituting the remaining Qs with Is.
The amino acid sequence (M_PRT525) set forth in SEQ ID NO: 37 is obtained by, with respect to Met-PRT410 (SEQ ID NO: 4), inserting two alanine residues in a region (A5) in which alanine residues are consecutive, and by deleting two domain sequences at the C-terminal side and substituting 13 glutamine residues (Q) with serine residues (S) or prolines (P) so that the molecular weight thereof is approximately the same as that of Met-PRT410.
The amino acid sequence (M_PRT699) set forth in SEQ ID NO: 38 is obtained by substituting all QQs in M PRT525 (SEQ ID NO: 37) with VLs.
The amino acid sequence (M PRT698) set forth in SEQ ID NO: 39 is obtained by substituting all QQs in M PRT525 (SEQ ID NO: 37) with VLs and substituting the remaining Qs with Is.
The glutamine residue content rate of any of the amino acid sequences 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: 38, and SEQ ID NO: 39 is 9% or less (Table 2).

TABLE 2

	Glutamine
	residue	GPGXX motif	Hydrophobicity
Modified fibroin	content rate	content rate	of REP

Met-PRT410	17.7%	27.9%	−1.52
(SEQ ID NO: 4)
M PRT888	6.3%	27.9%	−0.07
(SEQ ID NO: 27)
M_PRT965	0.0%	27.9%	−0.65
(SEQ ID NO: 28)
M_PRT889	0.0%	27.9%	0.35
(SEQ ID NO: 29)
M_PRT916	0.0%	27.9%	0.47
(SEQ ID NO: 30)
M_PRT918	0.0%	27.9%	0.45
(SEQ ID NO: 31)
M_PRT525	13.7%	26.4%	−1.24
(SEQ ID NO: 37)
M_PRT699	3.6%	26.4%	−0.78
(SEQ ID NO: 38)
M_PRT698	0.0%	26.4%	−0.03
(SEQ ID NO: 39)

The modified fibroin of (6-i) may consist of the 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: 38, or SEQ ID NO: 39.
The modified fibroin of (6-ii) includes an amino acid sequence having 90% or more sequence identity with the 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: 38, or SEQ ID NO: 39. The modified fibroin of (6-ii) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A). motif—REP]_m—(A)_nmotif. The sequence identity is preferably 95% or more.
The modified fibroin of (6-ii) preferably has the glutamine residue content rate of 9% or less. In addition, the modified fibroin of (6-ii) preferably has the GPGXX motif content rate of 10% or more.
The above-described modified fibroin may include a tag sequence at either or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect, and visualize the modified fibroin.
A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (6-iii) the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ ID NO: 41, or (6-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ ID NO: 41.
The amino acid sequences set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, and SEQ ID NO: 41 are respectively amino acid sequences obtained by adding the amino acid sequence (including a His tag and a hinge sequence) set forth in SEQ ID NO: 5 to the N-terminal of the amino acid sequences 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: 38, and SEQ ID NO: 39. Since only the tag sequence is added to the N-terminal, the glutamine residue content rate are not changed, and any of the amino acid sequences set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, and SEQ ID NO: 41 has the glutamine residue content rate of 9% or less (Table 3).

TABLE 3

	Glutamine
	residue	GPGXX motif	Hydrophobicity
Modified fibroin	content rate	content rate	of REP

PRT888	6.3%	27.9%	−0.07
(SEQ ID NO: 32)
PRT965	0.0%	27.9%	−0.65
(SEQ ID NO: 33)
PRT889	0.0%	27.9%	0.35
(SEQ ID NO: 34)
PRT916	0.0%	27.9%	0.47
(SEQ ID NO: 35)
PRT918	0.0%	27.9%	0.45
(SEQ ID NO: 36)
PRT699	3.6%	26.4%	−0.78
(SEQ ID NO: 40)
PRT698	0.0%	26.4%	−0.03
(SEQ ID NO: 41)

The modified fibroin of (6-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ ID NO: 41.
The modified fibroin of (6-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ ID NO: 41. The modified fibroin of (6-iv) is also a protein including a domain sequence represented by Formula 1: [(A)_nmotif—REP]_mor Formula 2: [(A). motif—REP]_m—(A)_nmotif. The sequence identity is preferably 95% or more.
The modified fibroin of (6-iv) preferably has the glutamine residue content rate of 9% or less. In addition, the modified fibroin of (6-iv) preferably has the GPGXX motif content rate of 10% or more.
The above-mentioned modified fibroin may include a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
(Manufacturing Method for Modified Fibroin)
The modified fibroin (hereinafter, also simply referred to as “protein”) according to the present embodiment can be produced, for example, by expressing a nucleic acid in a host transformed with an expression vector having the nucleic acid sequence encoding the modified fibroin and one or a plurality of regulatory sequences operably linked to the nucleic acid sequence.
The producing method for a nucleic acid encoding a modified fibroin is not particularly limited. For example, the nucleic acid is produced by cloning a gene encoding the natural fibroin by amplification with polymerase chain reaction (PCR) or the like and modifying the gene by a genetic engineering method, by chemically synthesizing the nucleic acid. The method for chemically synthesizing a nucleic acid is not particularly limited, and for example, the gene can be chemically synthesized by a method in which oligonucleotides are automatically synthesized by AKTA oligopilot plus 10/100 (GE Healthcare Japan Corporation) or the like and are linked by PCR or the like, based on the amino acid sequence information of the protein obtained from the NCBI web database or the like. In this case, in order to facilitate purification and/or confirmation of the protein, a nucleic acid may be synthesized such that a protein having an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon and a His10 tag to the N-terminal of the above amino acid sequence is encoded.
The regulatory sequence is a sequence (for example, a promoter, an enhancer, a ribosome binding sequence, or a transcription termination sequence) that controls the expression of a modified fibroin in a host, and can be appropriately selected depending on the type of the host. As a promoter, an inducible promoter that functions in a host cell and is capable of inducing the expression of a modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducer (an expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.
The type of the expression vector such as a plasmid vector, a viral vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector can be appropriately selected depending on the type of the host. As the expression vector, an expression vector that can autonomously replicate in a host cell or can be incorporated into a chromosome of a host and which contains a promoter at a position capable of transcribing the nucleic acid that encodes a protein is suitably used.
Both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used as a host.
Preferred examples of the prokaryotic host cells include bacteria belonging to the genus Escherichia, the genus Brevibacillus, the genus Serratia, the genus Bacillus, the genus Microbacterium, the genus Brevibacterium, the genus Corynebacterium, and the genus Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of the microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphilum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
In a case where a prokaryote is used as a host, examples of a vector into which a nucleic acid encoding a protein is introduced include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptll, pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569).
Examples of eukaryotic hosts include yeast and filamentous fungi (mold and the like). Examples of yeasts include yeasts belonging to the genus Saccharomyces, the genus Pichia, and the genus Schizosaccharomyces. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, and the genus Trichoderma.
In a case where a eukaryote is used as a host, examples of the vector into which a nucleic acid encoding a modified fibroin is introduced include YEp13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing an expression vector into the above host cell, any method can be used as long as the method introduces DNA into the host cell. Examples thereof include a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, and competent method.
As for the method for expressing a nucleic acid using a host transformed with an expression vector, secretory production, fusion protein expression, or the like, in addition to the direct expression, can be carried out according to the method described in Molecular Cloning, 2nd edition.
The modified fibroin can be produced, for example, by culturing a host transformed with the expression vector in a culture medium, producing and accumulating the protein in the culture medium, and then collecting the modified fibroin from the culture medium. The method for culturing a host in a culture medium can be carried out according to a method commonly used for culturing a host.
In the case where the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, any of a natural medium and a synthetic medium may be used as a culture medium of the host as long as the medium contains a carbon source, a nitrogen source, inorganic salts and the like which can be utilized by the host and the medium can be used for efficiently culturing the host.
As the carbon source, any carbon source that can be utilized by the transformed microorganism may be used. Examples of the carbon source that can be utilized include carbohydrates such as glucose, fructose, sucrose, and molasses, starch and starch hydrolyzates containing them, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source that can be utilized include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean cake and soybean cake hydrolyzate, and various fermented microbial cells and digested products thereof Examples of the inorganic salt that can be utilized include potassium dihydrogen phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
Culture of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15° C. to 40° C. The culture time is usually 16 hours to 7 days. It is preferable to maintain the pH of the culture medium during the culture at 3.0 to 9.0. 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, antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary during the culture. In a case of culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, in a case of culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used, and in a case of culturing a microorganism transformed with an expression vector using a trp promoter, indole acrylic acid or the like may be added to the medium.
The expressed protein can be isolated and purified by a commonly used method. For example, in a case where the protein is expressed in a dissolved state in cells, the host cells are recovered by centrifugation after the completion of the culture, suspended in an aqueous buffer solution, and then disrupted using an ultrasonicator, a French press, a Manton-Gaulin homogenizer, a Dyno-Mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified preparation can be obtained by a method commonly used for protein isolation and purification, that is, a solvent extraction method, a salting-out method using ammonium sulfate or the like, a desalting method, a precipitation method using an organic solvent, an anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Kasei Kogyo Kabushiki Kaisha), an cation exchange chromatography method using a resin such as S-Sepharose FF (manufactgured by Pharmacia Corporation), a hydrophobic chromatography method using a resin such as butyl sepharose or phenyl sepharose, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, or an electrophoresis method such as isoelectric focusing or the like, using the above methods singly or in combination thereof
In addition, in a case where the protein is expressed to form an insoluble body in the cell, similarly, the host cells are recovered, disrupted and centrifuged to recover the insoluble body of the protein as a precipitated fraction. The recovered insoluble body of the protein can be solubilized with a protein denaturing agent. After this operation, a purified preparation of the protein can be obtained by the same isolation and purification method as described above. In a case where the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
(Manufacturing Method for Artificial Fibroin Filament)
An artificial fibroin filament (hereinafter, sometimes referred to as “artificial protein filament” or simply “protein fiber”) may include other protein or a contaminant as long as it includes the modified fibroin as the main component. The artificial fibroin filament is preferably an artificial spider silk fibroin filament. The artificial fibroin filament can be manufactured by a known spinning method. That is, for example, in a case of manufacturing a protein filament including a modified fibroin as the main component, first, the modified fibroin produced according to the above-mentioned method is added to a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP), as necessary, together with an inorganic salt as a dissolution accelerator and dissolved to prepare a doping liquid. Then, using the doping liquid, spinning can be performed by a known spinning method such as wet-type spinning, dry-type spinning, dry-wet-type spinning, or melt spinning to obtain the protein filament. A preferred spinning method is wet-type spinning and dry-wet-type spinning
FIG. 3 is an explanatory view schematically illustrating one example of a spinning device for manufacturing a protein filament. A spinning device 10 shown in FIG. 3 is an example of a spinning device for dry-wet-type spinning and includes an extrusion device 1, an undrawn yarn manufacturing device 2, a wet heat drawing device 3, and a drying device 4.
A spinning method using the spinning device 10 will be described. First, a doping liquid 6 stored in a storage tank 7 is pushed out from a spinneret 9 by a gear pump 8. In the laboratory scale, the doping liquid may be filled in a cylinder and extruded from a nozzle using a syringe pump. Next, the extruded doping liquid 6 is supplied into a coagulation liquid 11 in a coagulation liquid bath 20 via an air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulate is formed. Then, the fibrous coagulate is supplied into a warm water 12 in a drawing bath 21 and is drawn. A drawing ratio is determined according to a speed ratio of a supply nip roller 13 to a withdrawing nip roller 14. Thereafter, the drawn fibrous coagulate is supplied to a drying device 4 and dried in a yarn path 22, and the protein filament 36 is obtained as a wound yarn body 5. Reference signs 18 a to 18 g indicate yarn guides.
The coagulation liquid 11 may be any solvent that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone. The coagulation liquid 11 may appropriately contain water. The temperature of the coagulation liquid 11 is preferably 0° 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, the extrusion speed is preferably 0.2 to 6.0 mL/hour per hole and more preferably 1.4 to 4.0 mL/hour. The distance that the coagulated modified fibroin passes through the coagulation liquid 11 (substantially, the distance from the yarn guide 18 a to the yarn guide 18 b) may be a length that allows efficient desolvation, for example, 200 to 500 mm. The withdrawing speed of the undrawn yarn may be, for example, 1 to 20 m/min and preferably 1 to 3 m/min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes and preferably 0.05 to 0.15 minutes. In addition, drawing (pre-drawing) may be performed in the coagulation liquid 11. The coagulation liquid bath 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 performed in a case of obtaining the protein filament, for example, a pre-drawing performed in the coagulation liquid bath 20 and a wet heat drawing performed in the drawing bath 21 are employed, and a dry heat drawing is also employed.
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 in heated steam. The 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, by 1 to 10 times and preferably by 2 to 8 times.
The dry heat drawing can be performed using an electric tubular furnace, a dry heat plate, or the like. The temperature may be, for example, 140° C. to 270° C. and preferably 160° C. to 230° C. In the dry heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, by 0.5 to 8 times and preferably by 1 to 4 times.
The wet heat drawing and the dry heat drawing may be performed independently or in combination or may be performed in multiple stages. That is, the wet heat drawing and the dry heat drawing can be performed in a suitable combination, for example, in a manner in which a first stage drawing is performed by wet heat drawing and a second stage drawing is performed by dry heat drawing or in a manner in which the first stage drawing is performed by wet heat drawing, the second stage drawing is performed by wet heat drawing, and a third stage drawing is performed by dry heat drawing.
The lower limit value of the final drawing ratio with respect to the undrawn yarn (or pre-drawn yarn) is preferably any 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, or 9 times or more, 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.
The length of the protein filament obtained by the above method can be suitably adjusted depending on the spinning conditions and is preferably more than 1,500 in, and may be 10,000 in or more, 15,000 in or more, or 20,000 in or more.
The artificial fibroin filament preferably has a shrinkage rate (shrinkage rate after drying) of more than 7% and may have a shrinkage rate of 10% or more, 15% or more, 25% or more, 32% or more, 40% or more, 48% or more, 56% or more, 64% or more, or 72% or more, when the artificial fibroin filament has been brought into contact with an aqueous medium to be described later (for example, water having a temperature lower than the boiling point of the water) and then dried. The shrinkage rate after drying is usually 80% or less. Here, the shrinkage rate after drying of the fibroin filament is defined by the following expression.
Shrinkage rate after drying={1−(length of artificial fibroin filament after being brought into contact with aqueous medium and subsequently dried/length of artificial fibroin filament before being brought into contact with aqueous medium)}×100 (%)
In addition, the artificial fibroin filament may have a shrinkage rate (shrinkage rate when wetted) of, for example, 2% or more and may have a shrinkage rate of 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, or 6% or more, when the artificial fibroin filament has been brought into a wet state by bringing the artificial fibroin filament into contact with an aqueous medium to be described later (for example, water having a temperature lower than the boiling point of the water). The upper limit of the shrinkage rate when wetted is not particularly limited but is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less. Here, the shrinkage rate when wetted can be calculated by the following expression.
Shrinkage rate when wetted={1−(length of artificial fibroin filament when wetted by being brought into contact with aqueous medium/length of artificial fibroin filament after being spun and before being brought into contact with aqueous medium)}×100 (%).
<Process b>
The process b (cutting process) is a process of cutting the artificial fibroin filament to obtain an artificial fibroin staple. Here, in a case where the process b is performed before process c, the artificial fibroin filament to be cut is an artificial fibroin filament before being crimped, and in a case where the process b is performed after the process c, the artificial fibroin filament to be cut is an artificial fibroin filament after being crimped.
The cutting can be performed using any device capable of cutting a protein fiber. An example of such a cutting device includes a tabletop-type fiber cutting machine (s/NO. IT-160201-NP-300).
The length of the staple is not particularly limited, but is preferably 20 mm or more, and may be 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.
<Process c>
The process c (crimping process) is a process of bringing the artificial fibroin filament or the artificial fibroin staple into contact with an aqueous medium and then crimping the artificial fibroin filament or the artificial fibroin staple (hereinafter sometimes referred to as “water crimping”). Here, in a case where the process c is performed before the process b, the artificial fibroin filament is crimped by being brought into contact with water to be curled, and in a case where the process c is performed after the process b, the artificial fibroin staple obtained by cutting the artificial fibroin filament is crimped by being brought into contact with water to be curled.
By being brought into contact with an aqueous medium, the artificial fibroin filament or the artificial fibroin staple can be crimped without depending on the external force. The aqueous medium is a medium of a liquid or gas (steam) containing water (including steam). The aqueous medium may be water or a mixed liquid of water and a hydrophilic solvent. Further, as the hydrophilic solvent, for example, a volatile solvent such as ethanol and methanol or a vapor thereof can be used. The aqueous medium may be a mixed liquid of water and a volatile solvent such as ethanol or methanol and is preferably water or a mixed liquid of water and ethanol. By using an aqueous medium containing a volatile solvent or a vapor thereof, it is possible to improve the drying speed after the water crimping, and further there is a possibility of imparting a soft texture to the crimped staple finally obtained. The ratio of water to the volatile solvent or the vapor thereof is not particularly limited, and, for example, water:volatile solvent or vapor thereof may be 10:90 to 90:10 by mass ratio. The proportion of water is preferably 30% by mass or more and may be 40% by mass or 50% by mass or more. In a case where the aqueous medium is a liquid, it is preferable to disperse an oil agent in the aqueous medium. In this case, the water crimping and the oil agent adhering can be performed at the same time. As the oil agent, any oil agent can be used as long it is a known oil agent used for general purposes including process passability and function impartability, such as an antistatic property, a friction reduction property, a flexibility imparting property, and a water repellency imparting property. The amount of the oil agent is not particularly limited and may be, for example, 1% to 10% by mass or 2% to 5% by mass with respect to the total amount of the oil agent and the aqueous medium.
The aqueous medium is preferably a liquid or gas containing water and having a temperature of 10° C. to 230° C. The 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, when the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably 100 to 230° C., more preferably 100 to 120° C. In a case where the steam of the aqueous medium is 230° C. or less, the heat denaturation of the 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 efficiently imparting crimpness, and is preferably 60° C. or lower from the viewpoint of highly maintaining fiber strength of the protein filament.
The time of bringing into contact with the aqueous medium is not particularly limited, but may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer, and preferably 10 minutes or shorter from the viewpoint of productivity. In the case of steam, it is considered that a high shrinkage rate can be obtained in a short time in comparison with a liquid. The contact with the aqueous medium may be carried out under normal pressure or under reduced pressure (for example, vacuum).
As a method for bringing into contact with the aqueous medium, a method of immersing an artificial fibroin filament or artificial fibroin staple in the aqueous medium, a method of spraying the steam of the aqueous medium to an artificial fibroin filament or artificial fibroin staple, and a method of exposing an artificial fibroin filament or an artificial fibroin staple to the environment filled with the steam of the aqueous medium. In a case where the aqueous medium is steam, bringing an artificial fibroin filament or an artificial fibroin staple into contact with the aqueous medium can be performed using a general steam setting device. Specific examples of the steam setting device include a device of product name: FMSA type steam setter (manufactured by Fukushin Kyougyo Co., Ltd.) and a device of a product name: EPS-400 (manufactured by Tsujii Dyeing Machine Manufacturing Co., Ltd.). The specific example of the method for crimping an artificial fibroin filament or an artificial fibroin staple by using the steam of the aqueous medium includes a method of accommodating an artificial fibroin filament or an artificial fibroin staple in a predetermined accommodating chamber and bringing the artificial fibroin filament or the artificial fibroin staple into contact with steam, while introducing the steam of the aqueous medium into the accommodating chamber and adjusting the temperature inside the accommodating chamber to the above-described predetermined temperature (for example, 100° C. to 230° C.).
The crimping process of an artificial fibroin filament or an artificial fibroin staple by contact with the aqueous medium is preferably performed in a state in which no tensile force is applied to the artificial fibroin filament or the artificial fibroin staple (not tensioned in the fiber axis direction) or in a state in which a predetermined amount of tensile force is applied (tensioned by a predetermined amount in the fiber axis direction). At this time, the degree of crimpness can be controlled by adjusting the tensile force applied to the artificial fibroin filament or the artificial fibroin staple. Examples of the adjusting method for the tensile force applied to the artificial fibroin filament or the artificial fibroin staple include, a method in which the artificial fibroin filaments or the artificial fibroin staples are, for example, hung by weighting units having various weights, and the loads applied to the filaments and staples are adjusted, a method in which both ends of the filament or staple are fixed in a state in which the filament or the staple are slackened, and the amount of slackness are changed variously, and a method in which the filament is wound around a wound body such as a paper tube or a bobbin so that the winding force is suitably changed (tightening force on the paper tube or a bobbin).
Further, the artificial fibroin filament or the artificial fibroin staple may be dried after being brought into contact with the aqueous medium (for example, water having a temperature lower than the boiling point of the water). The drying method is not particularly limited, and the drying may be natural drying, hot air drying, or hot roller drying. The drying temperature is not particularly limited and may be, for example, 20° C. to 150° C., preferably 40° C. to 120° C., and more preferably 60° C. to 100° C.
The artificial fibroin filament or the artificial fibroin staple after crimping and then drying preferably has a shrinkage rate (shrinkage rate after drying) of more than 7% and may have a shrinkage rate of 10% or more, 15% or more, 25% or more, 32% or more, 40% or more, 48% or more, 56% or more, 64% or more, or 72% or more. The shrinkage rate after drying is usually 80% or less. Here, the shrinkage rate after drying can be calculated by the following expression.
Shrinkage rate after drying={1−(length of artificial fibroin filament or artificial fibroin staple after crimping and then drying/length of artificial fibroin filament or artificial fibroin staple before being brought into contact with aqueous medium)}×100 (%)
In addition, the artificial fibroin filament or the artificial fibroin staple may have a shrinkage rate (shrinkage rate when wetted) of, for example, 2% or more and may have a shrinkage rate of 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, or 6% or more, when the artificial fibroin filament has been brought into a wet state by bringing the artificial fibroin filament into contact with an aqueous medium (or example, water having a temperature lower than the boiling point of the water). The upper limit of the shrinkage rate when wetted is not particularly limited but is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less. Here, the shrinkage rate when wetted can be calculated by the following expression.
Shrinkage rate when wetted={1−(length of artificial fibroin filament or artificial fibroin staple when wetted by being brought into contact with water/length of artificial fibroin filament after being spun and before being brought into contact with water)}×100 (%).
The process b and the process c may be performed in a batch system or a continuous system. In the case of a batch system, for example, the artificial fibroin staple obtained by cutting the artificial fibroin filament is put into a container containing an aqueous medium at an appropriate temperature, and after being brought into contact therewith for a certain time, and then taken out and dried. In the case of a continuous system, for example, while sending out the filament from a bobbin around which the artificial fibroin filament wound, after being immersed in an aqueous medium at an appropriate temperature, the filament is dried by blowing hot air or sending the filament out on a hot roller, and then cut continuously.
Use applications for protein crimped staple
The obtained protein crimped staple is a single fiber having a soft feel and can be used for manufacturing a composite material such as spun yarn, nonwoven fabric, and a composite.
A predetermined amount of the artificial fibroin fiber can be shrunk when first brought into contact with an aqueous medium after spinning Since the protein crimped staple obtained by the manufacturing method of the present invention has already been brought into contact with moisture (aqueous medium), it is possible to suppress the dimensional change (shrinkage) of the staple due to moisture absorption during storage after manufacturing the staple or during the manufacturing process for a product using the staple.

EXAMPLES

Hereinafter, the present invention will be described more specifically based on Examples. However, the present invention is not limited to the following Examples.
<Manufacturing example of artificial spider silk protein (artificial spider silk fibroin) filament>
(1) Preparation of Plasmid Expressing Strain
Based on the base sequence and the amino acid sequence of a fibroin (GenBank Accession No.: P46804.1, GI: 1174415) derived from Nephila clavipes, a modified fibroin having the amino acid sequence set forth in SEQ ID NO: 13 (hereinafter, also referred to as “PRT799”) was designed. The amino acid sequence set forth in SEQ ID NO: 13 has an amino acid sequence obtained by substituting, inserting, and deleting an amino acid residue for the purpose of improving productivity with respect to the amino acid sequence of the fibroin derived from Nephila clavipes, and furthermore, the amino acid sequence set forth in SEQ ID NO: 5 (tag sequence and hinge sequence) is added to the N-terminal of the sequence.
Next, a nucleic acid encoding PRT799 was synthesized. In the nucleic acid, an Ndel site was added to 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 enzymatically cleaved by treatment with Ndel and EcoRI, and then recombinated into a protein expression vector pET-22b (+) to obtain an expression vector.
(2) Expression of Protein
Escherichia coli BLR (DE3) was transformed with a 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 an LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium (Table 4) containing ampicillin so that the OD600 was 0.005. While maintaining the temperature of the culture solution at 30° C., flask culturing was carried out (for about 15 hours) until the OD600 reached 5, thereby obtaining a seed culture solution.

TABLE 4

Seed culture medium

	Reagent	Concentration (g/L)

	Glucose	5.0
	KH₂PO₄	4.0
	K₂HPO₄	9.3
	Yeast Extract	6.0
	Ampicillin	0.1

The seed culture solution was added to a jar fermenter containing 500 mL of a production medium (Table 5) so that the OD600 was 0.05. The culture was carried out while keeping the culture solution temperature at 37° C. and controlling the pH constant at 6.9. Further, the concentration of dissolved oxygen in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

TABLE 5

Production medium

	Reagent	Concentration (g/L)

	Glucose	12.0
	KH₂PO₄	9.0
	MgSO₄•7H₂O	2.4
	Yeast Extract	15
	FeSO₄•7H₂O	0.04
	MnSO₄•5H₂O	0.04
	CaCl₂•2H₂O	0.04
	GD-113 (anti-foaming agent)	0.1(mL/L)

Immediately after glucose in the production medium was completely consumed, a feed solution (455 g/lL of glucose and 120 g/lL of Yeast Extract) was added at a rate of 1 mL/min. The culture was carried out while keeping the culture solution temperature at 37° C. and controlling the pH constant at 6.9. Further, the concentration of dissolved oxygen in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Thereafter, 1 M isopropyl-P-thiogalactopyranoside
(IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the modified fibroin. 20 hours after the addition of IPTG, the culture solution was centrifuged to recover the bacterial cell pellet. SDS-PAGE was carried out using bacterial cell pellets prepared from the culture solution before the addition of IPTG and after the addition of IPTG, and the expression of the target modified fibroin was checked by the IPTG addition-dependent appearance of a band equivalent to the size of the target modified fibroin.
(3) Purification of Protein
The bacterial cell pellet recovered 2 hours after the addition of IPTG was washed with a 20 mM Tris-HCl buffer solution (pH 7.4). The bacterial cell pellet after washing was suspended in 20 mM Tris-HCl buffer solution (pH 7.4) containing about 1 mM PMSF, and the cell suspension was disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi SpA). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer solution (pH 7.4) until the obtained precipitate became highly pure. The precipitate after washing was suspended in 8 M guanidine buffer solution (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so that the concentration of the suspension was 100 mg/mL, and dissolved by stirring with a stirrer at 60° C. for 30 minutes. After dissolution, dialysis was carried out in water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white protein aggregate obtained after dialysis was recovered by centrifugation, the water content was removed with a lyophilizer, and the lyophilized powder was recovered to obtain the modified spider silk fibroin “PRT799”.
(4) Manufacturing of Protein Filament
The above-described modified fibroin (PRT799) was added to DMSO such that the concentration was 24% by mass, and then LiCl was added thereto as a dissolution accelerator such that the concentration was 4.0% by mass. Then, the modified fibroin was dissolved for 3 hours using a shaker to obtain a DMSO solution of the modified fibroin. Dust and bubbles in the obtained DMSO solution were removed to obtain a doping liquid. The solution viscosity of the doping liquid was 5,000 cP (centipoise) at 90° C.
Known dry-wet-type spinning was performed using the doping liquid obtained as described above and the spinning device 10 shown in FIG. 4, and the artificial spider silk fibroin fiber was wound around a bobbin. Here, the dry-wet-type spinning was performed under the following conditions.
Temperature of coagulation liquid (methanol): 5° C. to 10° C.
Drawing ratio: 4.52 times
Drying temperature: 80° C.

Example 1

An artificial spider silk protein staple was prepared by bundling a plurality of artificial spider silk filaments had been obtained in the manufacturing example of an artificial spider silk protein and had been wound around a bobbin, and by cutting the filament into a length of 40 mm by a table-top type fiber cutting machine. The prepared artificial spider silk protein staple was crimped by being immersed in water at 40° C. for 1 minute to be curled and then dried at 40° C. for 18 hours to obtain a crimped staple. The obtained crimped staple is shown in FIG. 5. The shrinkage rate of the artificial spider silk protein staple when immersed in water was 50%.

Example 2

An artificial spider silk protein staple was prepared by bundling a plurality of artificial spider silk filaments had been obtained in the manufacturing example of an artificial spider silk protein and had been wound around a bobbin, and by cutting the filament into a length of 40 mm by a table-top type fiber cutting machine. A commercially available antistatic oil agent was dispersed in water to a concentration of 1% by weight (concentration in the oil agent dispersion liquid) to obtain an oil agent dispersion liquid. The prepared artificial spider silk protein staple was crimped by being immersed in an oil agent dispersion liquid at 20° C. for 1 minute to be curled and then dried at 40° C. for 18 hours to obtain a crimped staple. The obtained crimped staple is shown in FIG. 6. The shrinkage rate of the artificial spider silk protein staple when immersed in the oil agent dispersion liquid was 50%.

Comparative Example

An artificial spider silk protein filament was simply cut to obtain an uncrimped staple. A photograph of the obtained uncrimped staple is shown in FIG. 7.

Example 3

An artificial spider silk protein staple was prepared by bundling a plurality of artificial spider silk filaments had been obtained in the manufacturing example of an artificial spider silk protein and had been wound around a bobbin, and by cutting the filament into a length of 40 mm by a table-top type fiber cutting machine. The prepared artificial spider silk protein staple was crimped by being immersed in a liquid mixture of water and methanol (50% by mass of a methanol concentration) at 20° C. for 1 minute to be curled and then dried at 40° C. for 18 hours to obtain a crimped staple.
The touch feeling of each of the crimped staple of Example 1 obtained by immersing in 100% water and the crimped staple of Example 3 obtained by immersing in the liquid mixture of water and methanol was confirmed by a sensitivity response test. It has been found that the crimped staple of Example 3 obtained by immersing in the liquid mixture of water and methanol has a softer feel.

REFERENCE SIGNS LIST

1 . . . extrusion device, 2 . . . undrawn yarn manufacturing device, 3 . . . wet heat drawing device, 4 . . . drying device, 6 . . . doping liquid, 10 . . . spinning device, 20 . . . coagulation liquid bath, 21 . . . drawing bath, 36 . . . protein filament.

Claims

1. A manufacturing method for a protein crimped staple, comprising:

a) preparing an artificial fibroin filament containing a modified fibroin;

b) cutting the artificial fibroin filament to obtain an artificial fibroin staple; and

c) performing crimping by bringing the artificial fibroin filament into contact with an aqueous medium to crimp the artificial fibroin filament before the cutting or bringing the artificial fibroin staple into contact with an aqueous medium to crimp the artificial fibroin staple after the cutting.

2. The manufacturing method for a protein crimped staple according to claim 1, wherein a shrinkage rate after drying of the artificial fibroin filament defined by the following expression is more than 7%,

shrinkage rate after drying={1−(length of artificial fibroin filament after being brought into contact with aqueous medium and subsequently dried/length of artificial fibroin filament before being brought into contact with aqueous medium)}×100 (%).

3. The manufacturing method for a protein crimped staple according to claim 1, wherein a shrinkage rate when wetted of the artificial fibroin filament defined by the following expression is 2% or more,

shrinkage rate when wetted={1−(length of artificial fibroin filament when wetted by being brought into contact with aqueous medium/length of artificial fibroin filament after being spun and before being brought into contact with aqueous medium)}×100 (%).

4. The manufacturing method for a protein crimped staple according to claim 1, wherein the modified fibroin is a modified spider silk fibroin, and the artificial fibroin filament is an artificial spider silk fibroin filament.

5. The manufacturing method for a protein crimped staple according to claim 1, wherein the aqueous medium used in the performing crimping is a liquid or gas containing water and having a temperature of 10° C. to 230° C.

6. The manufacturing method for a protein crimped staple according to claim 1, wherein the performing crimping further includes performing drying after the bringing the artificial fibroin filament or the artificial fibroin staple into contact with the aqueous medium.

7. The manufacturing method for a protein crimped staple according to claim 1, wherein the aqueous medium used in the performing crimping contains a volatile solvent.