JP7223984B2 - Method for producing protein spun yarn - Google Patents

Description

The present invention relates to a method for producing protein spun yarn.

Unlike synthetic fibers, protein fibers are biodegradable and require less energy for production and processing. Therefore, demand for protein fibers is expected to increase in various fields in response to the recent heightened awareness of environmental conservation. is

Filaments such as silk and staples such as wool are known as natural protein fibers. The former has a supple texture, while the latter has a soft feel and heat retention. there is

Recently, attempts have been made to process protein fibers and apply them to a wider range of applications. Methods have been proposed for making crimped yarns. Some studies have also been made on producing a protein crimped staple from protein filaments and using the protein crimped staple to obtain a spun yarn, a non-woven fabric, or the like.

As a method for obtaining a protein crimped spun yarn from protein filaments, for example, silk crimped filaments crimped by the crimp processing method disclosed in the above publication are cut to produce silk crimped staples, and the A method of spinning silk crimped staples is envisioned.

Japanese Patent Application Laid-Open No. 2006-207069 JP-A-9-119033

However, in the crimping method described in Patent Document 1, protein filaments are crimped by a mechanical processing method such as an indentation method. In the case of manufacturing, there is a problem that a dedicated crimping device is required and the processing cost is high. In addition, in the crimping method described in Patent Document 2, prior to the crimping process, it is necessary to perform a pretreatment to impart untwisted latent crimpability to natural silk, so this crimping method is used. Therefore, when producing spun yarn using a protein-crimped staple, there is a problem that the number of steps is increased, and there is a high possibility that the productivity will inevitably decrease.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for efficiently producing protein crimped spun yarn from protein filaments at low cost.

The present invention relates to, for example, the following inventions.
[1]
a) providing an artificial fibroin filament comprising modified fibroin;
b) a cutting step of cutting the artificial fibroin filament to obtain an artificial fibroin staple; obtaining an artificial fibroin crimped staple by performing a crimping step of bringing the fibroin staple into contact with an aqueous medium to crimp it;
c) a spinning step of spinning the artificial fibroin crimped staple;
A method for producing a protein yarn, comprising:
[2]
The method for producing a spun protein yarn according to [1], wherein the artificial fibroin filaments have a shrinkage rate of more than 7% due to crimping, as defined by the following formula.
Shrinkage ratio={1−(length of artificial fibroin filament after contact with aqueous medium/length of artificial fibroin filament before contact with aqueous medium)}×100 (%).
[3]
The method for producing a spun protein yarn according to [1] or [2], wherein the modified fibroin is modified spider silk fibroin and the artificial fibroin filament is an artificial spider silk fibroin filament.
[4]
The method for producing a protein spun yarn according to any one of [1] to [3], wherein the aqueous medium used in the crimping step is liquid or gas containing water at 10 to 230°C.
[5]
The method for producing a protein-spun yarn according to any one of [1] to [4], wherein the crimping step comprises contacting the artificial fibroin filament or the artificial fibroin staple with the aqueous medium and then drying the yarn.
[6]
The method for producing a protein spun yarn according to any one of [1] to [5], wherein the aqueous medium used in the crimping step contains a volatile solvent.

The production method of the protein spun yarn of the present invention employs a simple and unique crimping step of simply bringing the raw protein fibers into contact with an aqueous medium without using a dedicated crimping device. , the protein spun yarn can be produced easily, efficiently and at low cost.

FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an explanatory view schematically showing an example of a spinning device for producing protein filaments; 1 is a photograph of a protein spun yarn obtained in Example 1. FIG. 1 is a photograph of a protein spun yarn obtained in Example 1. FIG. 4 is a photograph of protein spun yarn obtained in Comparative Example.

A method for producing a crimped protein staple according to one embodiment of the present invention includes steps a, b and c described below.

<Step a>
The step is to prepare an artificial fibroin filament containing modified fibroin. Here, filaments (also called "long fibers") are obvious to those skilled in the art together with staples (also called "short fibers").

(modified fibroin)
The modified fibroin according to this embodiment has a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. protein containing The modified fibroin may have additional amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeated amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

As used herein, “modified fibroin” means artificially produced fibroin (artificial fibroin). The modified fibroin may be fibroin whose domain sequence differs from the amino acid sequence of naturally occurring fibroin, or may be fibroin whose amino acid sequence is identical to that of naturally occurring fibroin. As used herein, "naturally occurring fibroin" is also represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. is a protein containing a domain sequence that is

As long as the "modified fibroin" has the amino acid sequence specified in this embodiment, the amino acid sequence of naturally occurring fibroin may be used as it is. (For example, the amino acid sequence is modified by modifying the gene sequence of the cloned naturally-occurring fibroin), or it is artificially designed without relying on naturally-occurring fibroin. and synthetic ones (for example, those having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding a designed amino acid sequence).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically corresponding to the REP of the amino acid sequence). ) and is represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif means an array. Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and n is 2-27. n can be an integer from 2-20, 4-27, 4-20, 8-20, 10-20, 4-16, 8-16, or 10-16. In addition, (A) the ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed only of alanine residues. REP indicates an amino acid sequence composed of 2-200 amino acid residues. REP may be an amino acid sequence composed of 10-200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Modified fibroin is obtained by modifying the amino acid sequence by, for example, substituting, deleting, inserting and/or adding one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. can be obtained with Substitution, deletion, insertion and/or addition of amino acid residues can be performed by methods well known to those skilled in the art such as partial directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).

Naturally occurring fibroin is a protein comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. Specific examples include fibroin produced by insects or arachnids.

Examples of fibroin produced by insects include, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, and Pilosa ciliaceae. , Silk proteins produced by silkworms such as Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, hornet silk exhaled by larvae of wasps (Vespa simillima xantoptera) A protein is mentioned.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (nucleotide sequence), AAA27840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include, for example, spiders belonging to the genus Araneus such as Araneus spiders, Araneus spiders, Red spiders, Green spiders, and Beetle spiders; Spiders belonging to the genus Pronus, spiders belonging to the genus Pronus such as spiders belonging to the Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius such as the genus Ordgarius and the spiders belonging to the genus Ordgarius, spiders belonging to the genus Argiope such as the argiope spider, the argiope spider, and the argiope spider, and the like Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas, such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, and spiders Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as spiders belonging to the genus Cyclosa, spiders belonging to ), spiders belonging to the genus Cyclosa, and spiders belonging to the genus Chorizopes, such as spiders and spiders, Spiders belonging to the genus Tetragnatha such as the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucauge, such as the genus Leucauge, and the genus Nephila Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as golden spiders, spiders belonging to the genus Dyschiriognatha such as the brown spider, widow spiders such as the black widow spider, the redback spider, the gray widow spider and the western widow spider Spiders belonging to the genus Latrodectus and spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops Spider silk proteins can be mentioned. Spider silk proteins include, for example, MaSp (MaSp1 and MaSp2), dragline proteins such as ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence ), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (derived from Nephila clavata) No. AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidropin 2 [Euprosthenops australispin] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (nucleotide 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)), minor ampullate spidroin-like protein [Nephilengys crentata] (GenBank Accession and Section No. ABR37278.1 (amino acid sequence).

More specific examples of naturally occurring fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, among sequence information registered in NCBI GenBank, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION from among sequences containing INV as DIVISION. It can be confirmed by extracting the specific product character string from the sequence, CDS, and the sequence described with the specific character string from SOURCE to TISSUE TYPE.

Modified fibroin may be modified silk fibroin (modified silk protein amino acid sequence produced by silkworms), modified spider silk fibroin (modified spider silk protein amino acid sequence produced by spiders) thing). Among them, modified spider silk fibroin is preferably used.

Specific examples of modified fibroin include modified fibroin derived from major pit dragline protein produced in spider mammary gland, modified fibroin with reduced content of glycine residues, (A) _n motif Modified fibroin with reduced content, glycine residue content, and modified fibroin with reduced content of (A) _n motifs.

Modified fibroin derived from the major duct dragline protein produced in the greater vasculature of spiders includes proteins containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In the modified fibroin derived from the major pituitary dragline protein produced in the greater pituitary gland of spiders, in Formula 1, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, and 8 to 20. Integers are more preferred, integers from 10 to 20 are even more preferred, integers from 4 to 16 are even more preferred, integers from 8 to 16 are especially preferred, and integers from 10 to 16 are most preferred. In the modified fibroin derived from the major pit dragline silk protein produced in the greater pituitary gland of spiders, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues. More preferably ˜150 residues, even more preferably 20-100 residues, even more preferably 20-75 residues. Modified fibroin derived from the major pit dragline protein produced in the major pituitary gland of spiders is a glycine residue contained in the amino acid sequence represented by formula 1: [(A) _n motif-REP] _m , The total number of serine residues and alanine residues is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more of the total number of amino acid residues. .

A modified fibroin derived from the major duct dragline protein produced in the major pituitary gland of spiders comprises a unit of amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m and a C-terminal It may be a polypeptide whose sequence is the amino acid sequence shown in any of SEQ ID NOS: 14-16 or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOS: 14-16.

The amino acid sequence shown in SEQ ID NO: 14 is identical to the amino acid sequence consisting of the C-terminal 50 amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 15 has the sequence It is identical to the amino acid sequence shown in No. 14 with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID No. 16 has 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID No. 14. Identical to the amino acid sequence.

As a more specific example of the modified fibroin derived from the dragline silk protein produced in the major pituitary gland of spiders, (1-i) the amino acid sequence represented by SEQ ID NO: 17, or (1-ii) the sequence A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in number 17 can be mentioned. Preferably, the sequence identity is 95% or greater.

The amino acid sequence shown by SEQ ID NO: 17 is an amino acid sequence (SEQ ID NO: 18) consisting of an initiation codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus of ADF3. The 13th repeat region was increased to approximately double and mutated so that translation stops at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown by SEQ ID NO:17 is identical to the amino acid sequence shown by SEQ ID NO:16.

The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO:17.

A modified fibroin with reduced content of glycine residues has an amino acid sequence in which the domain sequence has a reduced content of glycine residues compared to naturally-occurring fibroin. The modified fibroin can be said to have an amino acid sequence corresponding to substitution of at least one or more glycine residues in REP with another amino acid residue, as compared with naturally occurring fibroin.

Modified fibroin with a reduced content of glycine residues has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, X represents an amino acid residue other than glycine.) In at least one motif sequence selected from, one glycine residue in at least one or more of the motif sequences is replaced with another amino acid residue. It may have an amino acid sequence.

In the modified fibroin with a reduced content of glycine residues, the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence. .

The modified fibroin with reduced content of glycine residues comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and is positioned most C-terminally from the domain sequence (A ) An amino acid sequence consisting of XGX (where G represents a glycine residue and X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the _n motif to the C-terminus of the above domain sequence Let z be the total number _of amino acid residues of the above domain sequence, and w , it may have an amino acid sequence with a z/w of 30% or more, 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

A modified fibroin with reduced content of glycine residues is produced by replacing one glycine residue in the GGX motif with another amino acid residue. It is preferable that the content ratio of the amino acid sequence consisting of XGX is increased. In the modified fibroin with reduced glycine residue content, the content of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, and 10% or less. It is more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z/w) of the amino acid sequence consisting of XGX below.

A method for calculating z/w will be described in more detail. First, in a fibroin (modified fibroin or naturally occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , the (A) _n located on the most C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50×3=150. In addition, for example, when there is an X (central X) contained in two XGX, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues be). w is the total number of amino acid residues contained in the domain sequence, excluding the sequence from the (A) _n motif positioned most on the C-terminal side to the C-terminus of the domain sequence. For example, for the domain sequence shown in Figure 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the most C-terminal (A) _n motif). Then z/w (%) can be calculated by dividing z by w.

In the modified fibroin with reduced glycine residue content, z/w is preferably 50.9% or more, more preferably 56.1% or more, and 58.7% or more. is more preferred, 70% or more is even more preferred, and 80% or more is even more preferred. Although the upper limit of z/w is not particularly limited, it may be, for example, 95% or less.

A modified fibroin with a reduced glycine residue content is obtained by, for example, substituting at least part of the nucleotide sequence encoding the glycine residue from the cloned naturally-derived fibroin gene sequence to encode another amino acid residue. It can be obtained by modifying to At this time, one glycine residue in the GGX motif and the 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, for example, it can be obtained by designing an amino acid sequence that satisfies the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the alteration corresponding to replacing the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , insertions and/or additions may be made to the amino acid sequence.

The other amino acid residue mentioned above is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( Hydrophobic amino acid residues such as M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W) residues, glutamine (Q) residues, asparagine (N) residues, serine (S ) residues, lysine (K) residues and glutamic acid (E) residues are preferred, and hydrophilic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

More specific examples of modified fibroin with reduced content of glycine residues include (2-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, or (2- ii) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12;

The modified fibroin of (2-i) will be explained. The amino acid sequence shown by SEQ ID NO: 3 is obtained by replacing all GGX in REP of the amino acid sequence shown by SEQ ID NO: 1 corresponding to naturally occurring fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 4 is obtained by deleting every two (A) _n motifs from the amino acid sequence shown by SEQ ID NO: 3 from the N-terminal side to the C-terminal side, and furthermore, in front of the C-terminal sequence. [(A) _n motif-REP] is inserted into the . The amino acid sequence shown by SEQ ID NO: 10 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown by SEQ ID NO: 4, and a part of glutamine (Q) residues. It is obtained by substituting serine (S) residues and deleting some amino acids on the N-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:4. The amino acid sequence shown by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). A His tag is added to the C-terminus of a sequence consisting of 4 repeats.

The z/w value in the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally occurring fibroin) is 46.8%. The z/w values of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 10, and the amino acid sequence represented by SEQ ID NO: 12 are each 58.7%, 70.1%, 66.1% and 70.0%. In addition, the x/y values of the amino acid sequences represented by SEQ ID NOs: 1, 3, 4, 10 and 12 at a jagged ratio (to be described later) of 1:1.8 to 11.3 are 15.0% and 15.0%, respectively. 0%, 93.4%, 92.7% and 89.3%.

The modified fibroin of (2-i) may consist of the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:12.

The modified fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:12. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and XGX contained in REP ( However, G represents a glycine residue and X represents an amino acid residue other than glycine. Sometimes it is preferred that z/w is 50.9% or greater.

The modified fibroin described above may contain a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

Examples of tag sequences include affinity tags that utilize specific affinity with other molecules. A specific example of the affinity tag is a histidine tag (His tag). His-tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has the property of specifically binding to metal ions such as nickel, so isolation of modified fibroin by metal chelating metal chromatography can be used for A specific example of the tag sequence is the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence containing a His tag).

Also, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.

Furthermore, an "epitope tag" using an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag, and the like. Modified fibroin can be easily purified with high specificity by using an epitope tag.

Furthermore, a tag sequence that can be cleaved by a specific protease can also be used. The modified fibroin from which the tag sequence has been cut off can also be recovered by treating the protein adsorbed via the tag sequence with protease.

More specific examples of modified fibroin containing a tag sequence include (2-iii) the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 8 , SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, and a modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more.

The amino acid sequences shown in SEQ ID NOs: 6, 7, 8, 9, 11 and 13 are added to the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 10 and 12, respectively, and the amino acid shown in SEQ ID NO: 5. A sequence (including a His-tag sequence and a hinge sequence) is added.

The modified fibroin of (2-iii) may consist of the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

The modified fibroin of (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and is contained in REP ( However, G represents a glycine residue and X represents an amino acid residue other than glycine. Sometimes it is preferred that z/w is 50.9% or greater.

The modified fibroin described above may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A modified fibroin with reduced content of (A) _n motifs has an amino acid sequence whose domain sequence has reduced content of (A) _n motifs compared to naturally-occurring fibroin. The domain sequence of the modified fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or a plurality of (A) _n motifs compared to the naturally occurring fibroin.

The modified fibroin with reduced content of the (A) _n motif may have an amino acid sequence corresponding to 10-40% deletion of the (A) _n motif from the naturally-occurring fibroin.

(A) Modified fibroin with reduced _n- motif content has at least 1 to 3 (A) _n -motifs from the N-terminal side to the C-terminal side in the domain sequence compared to naturally occurring fibroin. It may have an amino acid sequence corresponding to deletion of one (A) _n motif for each.

(A) Modified fibroin with reduced _n- motif content has a domain sequence that is at least two continuous from the N-terminal side to the C-terminal side compared to naturally-derived fibroin (A) _n- motif and deletion of one (A) _n motif repeated in this order.

(A) Modified fibroin with reduced _n- motif content has a domain sequence at least every two (A) _n -motifs deleted from the N-terminal side to the C-terminal side. It may have a sequence.

(A) Modified fibroin with reduced _n- motif content comprises a domain sequence represented by the formula 1: [(A) _n -motif-REP] _m , with adjacent The numbers of amino acid residues of the REPs of the two [(A) _n motif-REP] units that match are sequentially compared, and when the number of amino acid residues of the REP with a small number of amino acid residues is set to 1, the amino acid residue of the other REP is Let x be the maximum sum of the sums of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units with a cardinal number ratio of 1.8 to 11.3, and the total domain sequence It may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more, where y is the number of amino acid residues. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

A method for calculating x/y will be described in more detail with reference to FIG. FIG. 1 shows the domain sequence of the modified fibroin with the N-terminal and C-terminal sequences removed. The domain sequence is, from the N-terminal side (left side), (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called _n motif.

Two adjacent [(A) _n- motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, unselected [(A) _n motif-REP] units may be present. In FIG. 1, pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), pattern 4 (comparison of the first REP and A second REP comparison) was shown. Note that there are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two adjacent [(A) _n motif-REP] units selected is compared. Comparison is carried out by determining the ratio of the number of amino acid residues to the number of amino acid residues of the other. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the second REP is The ratio of amino acid residue numbers is 100/50=2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the fifth REP is 30/20=1.5.

In FIG. 1, a set of [(A) _n motif-REP] units having a ratio of 1.8 to 11.3 for the number of amino acid residues of the other is set to 1 for the one with the smaller number of amino acid residues. It is indicated by a solid line. Such a ratio is hereinafter referred to as a serration ratio. A set of [(A) _n motif-REP] units in which the other amino acid residue number ratio is less than 1.8 or greater than 11.3 when the number of amino acid residues is less than 1 is indicated by a dashed line. Indicated.

In each pattern, the numbers of all amino acid residues of two adjacent [(A) _n motif-REP] units indicated by solid lines are summed up (not only REP but also the number of amino acid residues of (A) _n motifs). be.). Then, the added total values are compared, and the total value (maximum total value) of the pattern with the maximum total value is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the largest.

Then x/y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

(A) In the modified fibroin in which _{the n-} motif content is reduced, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, It is even more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x /y is preferably 77.1% or more, and when the serration ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the serration ratio is 1 : 1.9 to 4.1, x/y is preferably 64.2% or more.

(A) Multiple modified fibroin with reduced _n -motif content are present in the domain sequence (A) When at least 7 _n -motifs are modified fibroin composed only of alanine residues, x/y is , preferably 46.4% or more, more preferably 50% or more, still more preferably 55% or more, even more preferably 60% or more, and 70% or more Even more preferably, it is particularly preferably 80% or more. The upper limit of x/y is not particularly limited as long as it is 100% or less.

(A) Modified fibroin with reduced _n- motif content encodes (A) _n- motif such that x/y is 64.2% or more from the cloned naturally-derived fibroin gene sequence. It can be obtained by deleting one or more of the sequences. Alternatively, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed such that x/y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence described above. In any case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are substituted, deleted, inserted and/or added. Alterations in the amino acid sequence corresponding to what has been done may be made.

(A) More specific examples of modified fibroin with reduced _n- motif content include (3-i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, or ( 3-ii) A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:12.

The modified fibroin of (3-i) will be explained. The amino acid sequence represented by SEQ ID NO: 2 is obtained by deleting every two (A) _n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin. , and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 4 is obtained by substituting all GGX in REP of the amino acid sequence shown by SEQ ID NO: 2 with GQX. The amino acid sequence shown by SEQ ID NO: 10 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown by SEQ ID NO: 4, and a part of glutamine (Q) residues. It is obtained by substituting serine (S) residues and deleting some amino acids on the N-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:4. The amino acid sequence shown by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). A His tag is added to the C-terminus of a sequence consisting of 4 repeats.

The x/y value of the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally-occurring fibroin) is 15.0% at the jagged ratio of 1:1.8 to 11.3. The x/y values in the amino acid sequence shown by SEQ ID NO: 2 and the amino acid sequence shown by SEQ ID NO: 4 are both 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 10 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 12 is 89.3%. The z/w values in the amino acid sequences represented by SEQ ID NOS: 1, 2, 4, 10 and 12 are 46.8%, 56.2%, 70.1%, 66.1% and 70.0%, respectively. be.

The modified fibroin of (3-i) may consist of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:12.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:12. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and from the N-terminal side to the C-terminal side , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (Giza ratio is 1:1.8 to 11.3) It is preferable that x/y is 64.2% or more, where x is the maximum sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.

The modified fibroin described above may contain the tag sequence described above at either or both of the N-terminus and C-terminus.

More specific examples of modified fibroin containing a tag sequence include (3-iii) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, and a modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more.

The amino acid sequences shown in SEQ ID NOs: 6, 7, 8, 9, 11 and 13 are added to the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 10 and 12, respectively, and the amino acid shown in SEQ ID NO: 5. A sequence (including a His tag) is added.

The modified fibroin of (3-iii) may consist of the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

The modified fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(3-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and from the N-terminal side to the C-terminal side , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Let x be the maximum value of the total sum of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. , x/y is preferably 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

The modified fibroin described above may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

Modified fibroin with reduced glycine residue content and (A) _n motif content has a domain sequence with reduced (A) _n motif content compared to naturally occurring fibroin In addition, it has an amino acid sequence with a reduced content of glycine residues. The domain sequence of the modified fibroin has at least one or more (A) _n motifs deleted, and at least one or more glycine residues in REP is replaced with another It can be said to have an amino acid sequence corresponding to the substitution of amino acid residues. That is, the modified fibroin has both the characteristics of the above-described modified fibroin with a reduced content of glycine residues and the modified fibroin with a reduced content of (A) _n motifs. Specific aspects are as described for modified fibroin with reduced content of glycine residues and (A) modified fibroin with reduced content of _n motifs.

As a more specific example of the modified fibroin with reduced glycine residue content and (A) _n- motif content, (4-i) the amino acid represented by SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12 (4-ii) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. Specific embodiments of the modified fibroin containing the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.

A modified fibroin according to another embodiment has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a large hydrophobicity index compared to naturally-derived fibroin, and / Or it may have an amino acid sequence that includes regions of high local hydrophobicity index corresponding to the insertion of one or more amino acid residues with high hydrophobicity index into REP.

A region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

The amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). A residue is more preferred.

In the modified fibroin according to this embodiment, one or more amino acid residues in REP are replaced with amino acid residues having a larger hydrophobicity index than in naturally occurring fibroin, and/or one Or in addition to the modification corresponding to the insertion of a plurality of amino acid residues with a large hydrophobicity index, further, one or more amino acid residues are substituted, deleted, inserted and / as compared to naturally occurring fibroin Alternatively, there may be a modification of the amino acid sequence corresponding to the addition.

For the modified fibroin according to the present embodiment, for example, one or more hydrophilic amino acid residues (for example, amino acid residues with a negative hydrophobicity index) in REP from the cloned naturally occurring fibroin gene sequence It can be obtained by substituting an amino acid residue (for example, an amino acid residue with a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid residues into REP. Further, for example, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acids in REP In addition to modifications corresponding to residue insertions, amino acid sequence modifications corresponding to substitutions, deletions, insertions and/or additions of one or more amino acid residues may also be made.

A modified fibroin according to still another embodiment comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and the (A) _n motif located on the most C-terminal side The total number of amino acid residues contained in a region where the average hydrophobic index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence up to the C-terminus from the domain sequence is p, and q is the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence, p It may have an amino acid sequence in which /q is 6.2% or more.

Hydropathy indexes of amino acid residues are known (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 157). 105-132). Specifically, the hydropathic index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

A method for calculating p/q will be described in more detail. For the calculation, the sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence (hereinafter referred to as “sequence A”) is used. First, in all REPs contained in sequence A, the average value of the hydrophobic index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to multiple "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

For example, if 20 “continuous 4 amino acid residues with an average hydrophobic index of 2.6 or more” are extracted (no overlap), the average hydrophobic index of 4 consecutive amino acid residues is 2 A region that is 0.6 or greater will contain 20 consecutive 4-amino acid residues (no overlap), and p is 20×4=80. Further, for example, when two "consecutive 4 amino acid residues having an average hydrophobicity index of 2.6 or more" exist overlapping by one amino acid residue, the hydrophobic index of the consecutive 4 amino acid residues A region with an average of 2.6 or more contains 7 amino acid residues (p=2×4−1=7, where “−1” is duplicate subtraction). For example, in the case of the domain sequence shown in FIG. 2, there are seven non-overlapping “continuous 4 amino acid residues with an average hydrophobicity index of 2.6 or more”, so p is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 2, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A) _n motif present at the end of the C-terminal side). Then p/q (%) can be calculated by dividing p by q. In the case of FIG. 2, 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, even more preferably 10% or more, and 20% or more. is even more preferred, and 30% or more is even more preferred. Although the upper limit of p/q is not particularly limited, it may be, for example, 45% or less.

For example, the modified fibroin according to the present embodiment is obtained by converting the amino acid sequence of the cloned naturally occurring fibroin into one or more hydrophilic amino acid residues (for example, hydrophobicity) in REP so as to satisfy the above p/q conditions. amino acid residues with a negative hydrophobicity index) with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index), and/or one or more hydrophobic amino acid residues in REP can be obtained by locally modifying the amino acid sequence containing a region with a large hydrophobicity index by inserting Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above p/q conditions from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more amino acid residues in REP have been replaced with amino acid residues having a higher hydrophobicity index compared to naturally-occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, further modification corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues may be performed. .

Amino acid residues with a large hydrophobicity index are not particularly limited, but areoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.

Another specific example of the modified fibroin is (5-i) the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23, or (5-ii) SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23 Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in .

The modified fibroin of (5-i) will be explained. The amino acid sequence represented by SEQ ID NO: 19 is obtained by deleting the amino acid sequence of consecutive alanine residues in the (A) _n motif of naturally occurring fibroin so that the number of consecutive alanine residues is five. . The amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 19 with two amino acid sequences (VLI) each consisting of 3 amino acid residues inserted every other REP, and represented by SEQ ID NO: 19. A part of amino acids on the N-terminal side is deleted so that the molecular weight is almost the same as that of the amino acid sequence obtained. The amino acid sequence shown by SEQ ID NO: 21 is the amino acid sequence shown by SEQ ID NO: 19, with two alanine residues inserted on the C-terminal side of each (A) _n motif, and a partial glutamine (Q) residue. A group is replaced with a serine (S) residue, and some amino acids on the N-terminal side are deleted so that the molecular weight is almost the same as that of the amino acid sequence shown in SEQ ID NO:19. The amino acid sequence shown by SEQ ID NO: 22 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 21. The amino acid sequence shown by SEQ ID NO: 23 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 21.

The modified fibroin of (5-i) may consist of the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:23.

The modified fibroin (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:23. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

The modified fibroin described above may contain a tag sequence at either or both of the N-terminus and C-terminus.

More specific examples of modified fibroin containing a tag sequence include (5-iii) the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24, SEQ ID NO: 25, or A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:26 can be mentioned.

The amino acid sequences represented by SEQ ID NOS: 24, 25 and 26 have the amino acid sequence represented by SEQ ID NO: 5 (including His tag sequence and hinge sequence) at the N-terminus of the amino acid sequences represented by SEQ ID NOS: 20, 22 and 23, respectively. It is an addition.

The modified fibroin of (5-iii) may consist of the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

The modified fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

The modified fibroin described above may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A modified fibroin according to yet another embodiment has an amino acid sequence with reduced content of glutamine residues compared to naturally-occurring fibroin.

The modified fibroin according to this embodiment preferably contains at least one motif selected from a GGX motif and a GPGXX motif in the amino acid sequence of REP.

When the modified fibroin according to this embodiment contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.

As used herein, the "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total by three (that is, the total number of G and P in the GPGXX motif) is defined as s, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is taken from the domain sequence. The GPGXX motif content rate is calculated as s/t, where t is the total number of amino acid residues in all REPs excluding (A) _n motifs.

In the calculation of the GPGXX motif content rate, the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is "the most C-terminal side (A) The sequence from _{the n} motif to the C-terminus of the domain sequence” (the sequence corresponding to REP) may include sequences that are poorly correlated with sequences characteristic of fibroin, and m is small If the domain sequence is short (that is, if the domain sequence is short), it affects the calculation result of the GPGXX motif content rate, so this effect is to be eliminated. When a "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA".

FIG. 3 is a schematic diagram showing the domain sequence of modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the modified fibroin domain sequence (“[(A) _n motif-REP] _m -(A) _n motif” type) shown in FIG. (A) The sequence obtained by removing the sequence from _{the n} motif to the C-terminus of the domain sequence from the domain sequence” (sequence shown as “region A” in FIG. 3). The number of GPGXX motifs is 7, and s is 7×3=21. Similarly, all REPs are "sequences obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" (the sequence shown as "region A" in FIG. .), the total number of amino acid residues t of all REPs further excluding the (A) _n motif from the sequence is 50+40+10+20+30=150. Next, s/t (%) can be calculated by dividing s by t, which is 21/150=14.0% for the modified fibroin in FIG.

The modified fibroin according to the present embodiment preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and preferably 0%. Especially preferred.

As used herein, "glutamine residue content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), all contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to “region A” in FIG. 3) REP, the total number of glutamine residues contained in the region is u, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and (A) _n The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues in all REPs excluding motifs. In the calculation of the glutamine residue content, the target is the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because of the reasons described above. It is the same.

The modified fibroin according to this embodiment has a domain sequence in which one or more glutamine residues in REP are deleted or replaced with other amino acid residues as compared with naturally-derived fibroin. It may have a corresponding amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than a glutamine residue, but preferably an amino acid residue with a higher hydrophobicity index than that of a glutamine residue. Hydrophobicity indexes of amino acid residues are shown in Table 1.

As shown in Table 1, amino acid residues having a higher hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H); can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

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, and 0.8. It is even more preferably 3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or less, or 0.7 or less.

As used herein, the "hydrophobicity of REP" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence (the sequence corresponding to "region A" in FIG. 1). In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is v, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues in all REPs excluding _n motifs. In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

The modified fibroin according to this embodiment has a domain sequence that lacks one or more glutamine residues in REP and/or one or more glutamine residues in REP compared to naturally occurring fibroin. In addition to alterations corresponding to substitutions of residues with other amino acid residues, there are further amino acid sequence alterations corresponding to substitutions, deletions, insertions and/or additions of one or more amino acid residues. good too.

The modified fibroin according to this embodiment can be produced, for example, by deleting one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence, and/or by deleting one or more glutamine residues in REP. can be obtained by substituting other amino acid residues. Also, for example, deletion of one or more glutamine residues in REP from the amino acid sequence of naturally occurring fibroin, and/or substitution of one or more glutamine residues in REP with other amino acid residues It can also be obtained by designing an amino acid sequence corresponding to , and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As a more specific example of the modified fibroin according to the present invention, (6-i) an amino acid represented by 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 modified fibroin comprising a sequence, or (6-ii) an amino acid sequence represented by 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 and 90% or more Modified fibroins comprising amino acid sequences with sequence identity can be mentioned.

The modified fibroin of (6-i) will be explained.

The amino acid sequence represented by SEQ ID NO: 4 (Met-PRT410) is based on the base and amino acid sequences of the naturally occurring fibroin Nephila clavipes (GenBank Accession No.: P46804.1, GI: 1174415), (A) _n Amino acid modifications were made to improve productivity, such as increasing the number of consecutive alanine residues to 5 in the amino acid sequence in which the alanine residues are consecutive in the motif. On the other hand, in Met-PRT410, since the glutamine residue (Q) is not modified, the glutamine residue content is comparable to that of naturally occurring fibroin.

The amino acid sequence (M_PRT888) shown in SEQ ID NO: 27 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VL.

The amino acid sequence (M_PRT965) shown in SEQ ID NO:28 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO:4) with TS and the remaining Q with A.

The amino acid sequence (M_PRT889) shown in SEQ ID NO: 29 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VL and the remaining Q with I.

The amino acid sequence (M_PRT916) shown in SEQ ID NO:30 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO:4) with VI and the remaining Q with L.

The amino acid sequence (M_PRT918) shown in SEQ ID NO:31 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO:4) with VF and the remaining Q with I.

The amino acid sequence (M_PRT525) shown in SEQ ID NO: 37 is the same as Met-PRT410 (SEQ ID NO: 4), by inserting two alanine residues into the region (A ₅ ) where the alanine residues are continuous, and increasing the molecular weight of Met-PRT410. , two C-terminal domain sequences are deleted and 13 glutamine residues (Q) are replaced with serine residues (S) or proline residues (P) so as to be almost the same as .

The amino acid sequence (M_PRT699) represented by SEQ ID NO: 38 is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 37) with VL.

The amino acid sequence (M_PRT698) represented by SEQ ID NO: 39 is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 37) with VL, and replacing the remaining Q with I.

The amino acid sequences represented by 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 all have a glutamine residue content of 9% or less (Table 2 ).

The modified fibroin of (6-i) may consist of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 17 .

(6-ii) modified fibroin has 90% or more sequence identity with the amino acid sequence shown 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 and an amino acid sequence having The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.

The modified fibroin described above may contain a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

More specific examples of modified fibroin containing a tag sequence include (6-iii) the amino acid represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, or SEQ ID NO: 20 A modified fibroin comprising a sequence, or (6-iv) an amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 20 and 90% or more Modified fibroins comprising amino acid sequences with sequence identity can be mentioned.

The amino acid sequences shown 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 SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively. , SEQ ID NO: 31, SEQ ID NO: 38 and SEQ ID NO: 39 to which the amino acid sequence of SEQ ID NO: 5 (including His tag sequence and hinge sequence) is added to the N-terminus. Since the tag sequence was only added to the N-terminus, there was no change in the glutamine residue content, 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. All of the amino acid sequences represented by have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) may consist of the amino acid sequence shown 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 .

(6-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown 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 and an amino acid sequence having The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

The modified fibroin described above may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

(Method for producing modified fibroin)
Modified fibroin according to the present embodiment (hereinafter sometimes simply referred to as "protein") includes, for example, a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence can be produced by expressing the nucleic acid in a host transformed with an expression vector having

A method for producing a nucleic acid encoding modified fibroin is not particularly limited. For example, using a gene encoding natural fibroin, amplifying and cloning by polymerase chain reaction (PCR), etc., modifying by genetic engineering method, or chemically synthesizing the nucleic acid. can do. The method for chemically synthesizing the nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of the protein obtained from the NCBI web database, etc., with AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.). A gene can be chemically synthesized by a method of linking automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and/or confirmation of the protein, a nucleic acid encoding a protein consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is synthesized. good too.

The regulatory sequence is a sequence (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) that controls expression of the modified fibroin in the host, and can be appropriately selected according to the type of host. As a promoter, an inducible promoter that functions in host cells and can induce the expression of modified fibroin may be used. An inducible promoter is a promoter whose transcription can be controlled by physical factors such as the presence of an inducer (expression inducer), the absence of a repressor molecule, or an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected from plasmid vectors, virus vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, etc., depending on the type of host. Expression vectors that are capable of autonomous replication in host cells or that are capable of integration into host chromosomes and that contain a promoter at a position at which a nucleic acid encoding a protein can be transcribed are preferably used.

As hosts, both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. 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 ammonium philum. Microorganisms belonging to the genus Brevibacterium include, for example, Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

When prokaryotes are used as hosts, examples of vectors for introducing nucleic acids encoding proteins include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2. (Japanese Unexamined Patent Application Publication No. 2002-238569).

Eukaryotic hosts include, for example, yeast and filamentous fungi (such as molds). Examples of yeast include yeast belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces, and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding modified fibroin is introduced include YEp13 (ATCC37115), YEp24 (ATCC37051), and the like. Any method for introducing DNA into the host cell can be used as the method for introducing the expression vector into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like.

As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning, Second Edition. .

Modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in the culture medium can be carried out according to a method commonly used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the host so that the host can be efficiently cultured. Either a natural medium or a synthetic medium may be used as long as the medium is used.

Any carbon source can be used as long as it can be assimilated by the above-mentioned transformed microorganism. Examples include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch and starch hydrolysates, acetic acid and propionic acid, and the like. Organic acids and alcohols such as ethanol and propanol can be used. Nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermented cells and their digests can be used. Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

Prokaryotes such as E. coli or eukaryotes such as yeast can be cultured under aerobic conditions such as shaking culture or deep aeration stir culture. The culture temperature is, for example, 15-40°C. Culture time is usually 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture, if necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using a lac promoter, and indole acrylic when culturing a microorganism transformed with an expression vector using a trp promoter. Acids and the like may be added to the medium.

Isolation and purification of the expressed protein can be performed by commonly used methods. For example, when the protein is expressed in a dissolved state in cells, the host cells are recovered by centrifugation after the completion of culture, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, or a mantongaurin. The host cells are disrupted with a homogenizer, a dyno mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for protein isolation and purification, that is, a solvent extraction method, a salting-out method with ammonium sulfate, a desalting method, an organic solvent Precipitation method, diethylaminoethyl (DEAE)-Sepharose, anion exchange chromatography method using resins such as DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), positive chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing A purified sample can be obtained by using the methods such as the above alone or in combination.

In addition, when the protein forms an insoluble form in the cells and is expressed, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble form of the protein as a precipitate fraction. The collected insoluble protein can be solubilized with a protein denaturant. After this operation, a purified sample of protein can be obtained by the same isolation and purification method as described above. When the protein is extracellularly secreted, 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 sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.

(Manufacturing method of artificial fibroin filament)
Artificial fibroin filaments (hereinafter sometimes referred to as "artificial protein filaments" or simply "protein fibers") contain artificial spider silk fibroin as a main component, and may contain other proteins and contaminants. Artificial fibroin filaments can be produced by known spinning methods. That is, for example, when producing a protein filament containing modified fibroin as a main component, first, the modified fibroin produced according to the above-described method is treated with dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), and formic acid. , or a solvent such as hexafluoroisopropanol (HFIP), if necessary, it is added together with an inorganic salt as a dissolution accelerator and dissolved to prepare a dope solution. Then, using this dope solution, spinning is performed by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning to obtain protein filaments. Preferred spinning methods include wet spinning or dry-wet spinning.

FIG. 3 is an explanatory diagram schematically showing an example of a spinning apparatus for producing protein filaments. A spinning device 10 shown in FIG. 3 is an example of a spinning device for dry-wet 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 apparatus 10 will be described. First, the dope liquid 6 stored in the storage tank 7 is pushed out from the mouthpiece 9 by the gear pump 8 . On a lab scale, the dope solution may be filled into a cylinder and pushed out through a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied through the air gap 19 into the coagulation liquid 11 of the coagulation liquid bath 20, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulum is formed. The fibrous coagulum is then fed into the hot water 12 in the drawing bath 21 and drawn. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14 . After that, the drawn fibrous coagulum is supplied to the drying device 4 and dried in the thread guide 22 to obtain the protein filaments 36 as the wound body 5 . 18a-18g are thread guides.

The coagulating liquid 11 may be any solvent that can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulation liquid 11 may contain water as appropriate. The temperature of the coagulation liquid 11 is preferably 0 to 30.degree. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the mouthpiece 9, the extrusion rate is preferably 0.2 to 6.0 mL/hour, 1.4 to 4.0 mL/hour per hole. Time is more preferred. The distance over which the coagulated modified fibroin passes through the coagulation liquid 11 (substantially, the distance from the thread guide 18a to the thread guide 18b) is sufficient as long as the solvent can be removed efficiently. 500 mm. The undrawn yarn take-up speed may be, for example, 1 to 20 m/min, preferably 1 to 3 m/min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11 . The coagulation liquid bath 20 may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage as required.

In addition to the pre-stretching performed in the coagulation bath 20 and the wet-heat stretching performed in the stretching bath 21, dry-heat stretching is employed as the stretching performed when obtaining the protein filaments.

The wet heat stretching can be carried out in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating. The temperature may be, for example, 50 to 90°C, preferably 75 to 85°C. In wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, preferably 2 to 8 times.

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, preferably 160°C to 230°C. In the dry heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, preferably 1 to 4 times.

The wet heat stretching and the dry heat stretching may be carried out individually, or they may be carried out in multiple stages or in combination. That is, the first step drawing is performed by wet heat drawing, the second step drawing is performed by dry heat drawing, or the first step drawing is performed by wet heat drawing, the second step drawing is performed by wet heat drawing, and the third step drawing is performed by dry heat drawing. For example, wet heat stretching and dry heat stretching can be combined as appropriate.

The lower limit of the final draw ratio is preferably more than 1 times, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times with respect to the undrawn yarn (or pre-drawn yarn). 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less. , 12 times or less, 11 times or less, and 10 times or less.

The length of the protein filament obtained by the above method can be appropriately adjusted depending on the spinning conditions, and preferably exceeds 1500 m, and may be 10000 m or longer, 15000 m or longer, or 20000 m or longer.

The artificial fibroin filament preferably has a shrinkage rate of more than 7% when contacted with an aqueous medium described later, and is 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. Shrinkage is usually 80% or less. Here, the shrinkage rate of fibroin filaments is defined by the following equation.
Shrinkage ratio = {1-(length of fibroin filament after contact with aqueous medium/length of fibroin filament before contact with aqueous medium)} x 100 (%)

<Step b>
The step b includes a cutting step of cutting the artificial fibroin filament to obtain an artificial fibroin staple, and prior to the cutting step, the artificial fibroin filament is brought into contact with an aqueous medium and crimped, or after the cutting step, the artificial fibroin staple is obtained. is brought into contact with an aqueous medium to crimp to obtain an artificial fibroin crimped staple. That is, step b includes a cutting step and a crimping step, and the crimping step may be performed before the cutting step or after the cutting step. When the cutting step is performed before the crimping step, the artificial fibroin filament before being crimped is cut, and the artificial fibroin staple obtained by cutting is crimped. When the cutting step is performed after the crimping step, it is the artificial fibroin filament to be crimped and the artificial fibroin filament to be cut after being crimped.

The cutting step can be performed using any device capable of cutting protein fibers. Such a device includes, for example, a desktop fiber cutting machine (s/NO.IT-160201-NP-300).

Although the length of the staple is not particularly limited, it is preferably 20 mm or more, and may be 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.

The crimping step is a step of bringing artificial fibroin filaments or artificial fibroin staples into contact with an aqueous medium to crimp them (hereinafter sometimes referred to as "water crimping").

The artificial fibroin filament or the artificial fibroin staple can be crimped without external force by bringing it into contact with the aqueous medium. An aqueous medium is a liquid or gaseous (steam) medium containing water (including water vapor). The aqueous medium may be water or a mixture of water and a hydrophilic solvent. Volatile solvents such as ethanol and methanol or vapors thereof can also be used as the hydrophilic solvent. The aqueous medium may be a liquid mixture of water and a volatile solvent such as ethanol or methanol, preferably water or a liquid mixture of water and ethanol. By using an aqueous medium containing a volatile solvent or its vapor, it is possible to improve the drying speed after water crimping, and further to impart a soft texture to the finally obtained crimped staple. There is The ratio of water to the volatile solvent or its vapor is not particularly limited, and for example, the weight ratio of water:volatile solvent or its vapor may be from 10:90 to 90:10. The proportion of water is preferably 30% by mass or more, and may be 40% by mass or 50% by mass or more. When the aqueous medium is liquid, it is preferable to disperse an oil agent in the aqueous medium. In this case, water crimping and oil adhesion can be performed simultaneously. As the oil agent, for example, any known oil agent used for general purposes such as antistatic, friction reducing, flexibility imparting, or water repellency imparting process passability and functional imparting. Either can be used. 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 (including water vapor) at a temperature of 10 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 gas (steam), the temperature of the aqueous medium is preferably 100 to 230°C, more preferably 100 to 120°C. When the steam of the aqueous medium is 230° C. or less, heat denaturation of protein filaments can be prevented. When the aqueous medium is 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 crimping. 60° C. or less is preferable.

The contact time 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. Also, in the case of steam, it is considered that a large shrinkage rate can be obtained in a shorter time than in the case of liquid. The contact with the aqueous medium may be carried out under normal pressure or under reduced pressure (for example, vacuum).

The method of contacting with the aqueous medium includes a method of immersing the artificial fibroin filament or the artificial fibroin staple in the aqueous medium, a method of spraying the artificial fibroin filament or the artificial fibroin staple with the steam of the aqueous medium, and a method of filling the steam of the aqueous medium. Examples include a method of exposing the artificial fibroin filament or artificial fibroin staple to the environment. When the aqueous medium is steam, contacting the artificial fibroin filaments or artificial fibroin staples with the aqueous medium can be done using a common steam set device. Specific examples of the steam set device include devices such as product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and product name: EPS-400 (manufactured by Tsujii Senki Kogyo Co., Ltd.). As a specific example of the method of crimping artificial fibroin filaments or artificial fibroin staples with steam of an aqueous medium, the artificial fibroin filaments or artificial fibroin staples are accommodated in a predetermined storage chamber, and steam of an aqueous medium is introduced into the storage chamber. Then, the artificial fibroin filament or the artificial fibroin staple is brought into contact with the steam while adjusting the temperature inside the storage chamber to the predetermined temperature (for example, 100° C. to 230° C.).

In the process of crimping the artificial fibroin filaments or artificial fibroin staples by contact with an aqueous medium, preferably no tensile force is applied to the single fibers or bundles (such as skeins) of the artificial fibroin filaments or artificial fibroin staples ( It is performed in a state in which no tension is applied in the fiber axis direction) or in a state in which a predetermined amount is applied (a predetermined amount of tension is applied in the fiber axis direction). By adjusting the tensile force applied to the artificial fibroin filament or artificial fibroin staple at that time, it is possible to control the degree of crimping. As a method for adjusting the tensile force applied to the artificial fibroin filament or the artificial fibroin staple, for example, by suspending weights of various weights on the artificial fibroin filament or the artificial fibroin staple, the fibroin fibroin or the artificial fibroin staple is loaded. A method of adjusting the load, a method of fixing both ends of the filament or staple in a slackened state and varying the amount of slackness, a method of winding the filament around an object to be wound such as a paper tube or a bobbin, and at that time A method of appropriately changing the winding force (clamping force to the paper tube or bobbin) can be used.

After contacting the artificial fibroin filaments or artificial fibroin staples with the aqueous medium, they may be further dried. The drying method is not particularly limited, and drying may be natural drying or drying with hot air or a hot roller. The drying temperature is not particularly limited, and may be, for example, 20 to 150°C, preferably 40 to 120°C, more preferably 60 to 100°C.

The shrinkage ratio of the artificial fibroin filament or artificial fibroin staple after crimping is preferably more than 7%, 10% or more, 15% or more, 25% or more, 32% or more, 40% or more, It may be 48% or more, 56% or more, 64% or more, or 72% or more. Shrinkage is usually 80% or less. Here, the shrinkage rate can be calculated by the following formula.
Shrinkage ratio = {1-(length of artificial fibroin filament or artificial fibroin staple after crimping process/length of artificial fibroin filament or artificial fibroin staple before crimping process)} x 100 (%)

The cutting process and the crimping process may be performed batchwise or continuously. In the case of a batch method, for example, artificial fibroin staples obtained by cutting artificial fibroin filaments are put into a container containing an aqueous solvent at an appropriate temperature, brought into contact with each other for a certain period of time, then taken out and dried. . In the case of the continuous type, for example, while sending out the artificial fibroin filament from a bobbin, the filament is immersed in an aqueous medium at an appropriate temperature, then dried by blowing hot air or sending it out on a hot roller, and then Cut continuously.

The protein-crimped staple obtained by step b is a monofilament that exhibits a soft feel and can be used in the production of composite materials such as spun yarns, non-woven fabrics and composites.

Artificial fibroin fibers may shrink by a certain amount upon initial contact with an aqueous medium after spinning. Since the protein-crimped staple obtained by the production method of the present invention has already been contacted with water (aqueous solvent), moisture absorption during storage after staple production or during the production process of products using staples does not occur. It is possible to suppress the dimensional change (shrinkage) of the staple due to

<Step c>
Step c is a spinning step of spinning the artificial fibroin crimped staple obtained above. The spinning method is not particularly limited, and any known spinning method can be used. For example, methods such as cotton spinning, worsted, and woolen methods can be mentioned, and the woolen method is preferred from the viewpoint of swelling, which is the effect of crimping. Machines used for these spinning methods are not particularly limited, and machines that are commonly used may be used. Moreover, in the spinning process, it is preferable to first open and defibrate the artificial fibroin crimped staple with an opener, a breaker, or the like.

Types of spun yarn include, for example, spun yarn of 100% artificial fibroin, blended yarn of crimped staple of 100% artificial fibroin and other protein crimped staple or chemical fiber crimped staple, artificial fibroin and other components. and a blended yarn of a crimped staple containing artificial fibroin and other ingredients and another protein crimped staple or a chemical fiber crimped staple.

In order to make the artificial fibroin crimped staples easier to unravel, it is preferable to apply an oil solution in advance. Oil agent adhesion can be carried out at any stage in the manufacturing process. For example, before the crimping process, at the same time as the crimping process, or after the crimping process, the oil agent may be applied. The oil agent is not particularly limited, and any known oil agent that is used for general purposes such as antistatic, friction reducing, flexibility imparting, or water repellency imparting process passability and functionality. Either can be used.

The spun protein yarn obtained by the method according to the present invention exhibits a relatively soft feel because it contains protein-crimped staples having a soft feel. In addition, since it is in contact with moisture (aqueous medium), the dimensional change ( contraction) can be suppressed.

EXAMPLES The present invention will now be described more specifically based on examples. However, the present invention is not limited to the following examples.

<Production example of artificial spider silk protein (artificial spider silk fibroin) filament>
(1) Preparation of Plasmid Expression Strain Based on the nucleotide sequence and amino acid sequence of Nephila clavipes-derived fibroin (GenBank Accession No.: P46804.1, GI: 1174415), the amino acid sequence represented by SEQ ID NO: 13 was prepared. A modified fibroin (hereinafter also referred to as “PRT799”) having The amino acid sequence represented by SEQ ID NO: 13 has an amino acid sequence obtained by subjecting the amino acid sequence of fibroin derived from Nephila clavipes to substitution, insertion and deletion of amino acid residues for the purpose of improving productivity. , and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 5 is added to the N-terminus.

Next, a nucleic acid encoding PRT799 was synthesized. The nucleic acid was added with an NdeI site at the 5' end and an EcoRI site downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with restriction enzymes NdeI and EcoRI, excised, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Protein Expression Escherichia coli BLR(DE3) was transformed with the pET22b(+) expression vector containing the nucleic acid encoding the protein having the amino acid sequence shown in SEQ ID NO:13. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin to an OD ₆₀₀ of 0.005. The temperature of the culture solution was kept at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) to an OD ₆₀₀ of 0.05. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and culture was carried out for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the modified fibroin. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of the desired modified fibroin size band depending on the addition of IPTG. bottom.

(3) Purification of Protein Cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The precipitate after washing was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH 7.0) to a concentration of 100mg/mL, and was incubated at 60°C for 30 minutes. Stir with a stirrer for 1 minute to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed with a freeze dryer, and the freeze-dried powder was recovered to obtain modified spider silk fibroin "PRT799".

(4) Production of Protein Filaments After adding the modified fibroin (PRT799) described above to DMSO at a concentration of 24% by mass, LiCl was added as a dissolution accelerator to a concentration of 4.0% by mass. Then, using a shaker, the modified fibroin was dissolved for 3 hours to obtain a DMSO solution. Dust and bubbles in the obtained DMSO solution were removed to obtain a dope solution. The solution viscosity of the dope solution was 5000 cP (centipoise) at 90°C.

Using the dope solution obtained as described above and the spinning apparatus 10 shown in FIG. 4, known dry-wet spinning was performed to wind the artificial spider fibroin fiber around a bobbin. Here, dry-wet spinning was performed under the following conditions.
Temperature of coagulation liquid (methanol): 5-10°C
Stretch ratio: 4.52 times Drying temperature: 80°C

<Example 1>
Artificial spider silk protein staples were produced by bundling a plurality of artificial spider silk filaments obtained in the production example of artificial spider silk protein and wound on a bobbin and cutting them to an average length of 40 mm with a desktop fiber cutting machine. . The produced artificial spider silk protein staple was immersed in water at 40° C. for 1 minute to crimp to crimp, and then dried at 40° C. for 18 hours to obtain a crimped staple. The obtained crimped staple was spun by a known spinning device. The obtained spun yarn was a single yarn having a yarn count (hair count) of about 18.5 Nm. A photograph of the spun yarn is shown in FIG.

<Example 2>
Artificial spider silk protein staples were produced by bundling a plurality of artificial spider silk filaments obtained in the production example of artificial spider silk protein and wound on a bobbin and cutting them to an average length of 40 mm with a desktop fiber cutting machine. . A commercially available antistatic oil was dispersed in water to a concentration of 1% by weight to obtain an oil dispersion. The produced artificial spider silk protein staple was immersed in an oil dispersion at 20° C. for 1 minute to crimp and crimped, and then dried at 40° C. for 18 hours to obtain a crimped staple. The obtained crimped staple was spun by a known spinning device. The obtained spun yarn was a single yarn having a yarn count (hair count) of about 18.5 Nm. A photograph of the spun yarn is shown in FIG.

<Comparative example>
The artificial spider silk protein filaments were simply cut to obtain crimp-free staples. A photograph of the obtained uncrimped staple is shown in FIG.

<Example 3>
Artificial spider silk protein staples were produced by bundling a plurality of artificial spider silk filaments obtained in the production example of artificial spider silk protein and wound on a bobbin and cutting them to an average length of 40 mm with a desktop fiber cutting machine. . The prepared artificial spider silk protein staple is crimped by immersing it in a mixture of water and methanol (methanol concentration: 50% by mass) at 20°C for 1 minute to crimp it, and then drying it at 40°C for 18 hours. , to obtain a crimped staple. The obtained crimped staple was spun by a known spinning device. The obtained spun yarn was a single yarn having a yarn count (hair count) of about 18.5 Nm.

The feel of the spun yarn of Example 1 obtained by immersing it in 100% water and the spun yarn of Example 3 obtained by immersing it in a mixture of water and methanol were confirmed by sensory tests. It was found that the spun yarn of Example 3 obtained by soaking in a mixture of water and methanol had a softer feel. It was suggested that hardening of fibers could be suppressed by adding an oil agent.

DESCRIPTION OF SYMBOLS 1... Extrusion apparatus 2... Undrawn yarn manufacturing apparatus 3... Wet heat drawing apparatus 4... Drying apparatus 6... Dope solution 10... Spinning apparatus 20... Solidification liquid tank 21... Drawing bath 36... Protein filament.

Claims (6)

a) providing an artificial fibroin filament comprising modified fibroin (excluding naturally occurring fibroin and fibroin having the same amino acid sequence as naturally occurring fibroin) ;
b) a cutting step of cutting the artificial fibroin filament to obtain an artificial fibroin staple; obtaining an artificial fibroin crimped staple by performing a crimping step of bringing the fibroin staple into contact with an aqueous medium to crimp it;
c) a spinning step of spinning the artificial fibroin crimped staple;
including
A method for producing a spun protein yarn, wherein the aqueous medium used in the crimping step is water at 10 to 100° C. or a liquid mixture of water and a volatile solvent (excluding gas). 2. The method for producing a protein spun yarn according to claim 1, wherein the artificial fibroin filament has a shrinkage rate of more than 7% due to crimping defined by the following formula.
Shrinkage ratio={1−(length of artificial fibroin filament after contact with aqueous medium/length of artificial fibroin filament before contact with aqueous medium)}×100 (%). The method for producing a protein spun yarn according to claim 1 or 2, wherein the modified fibroin is modified spider silk fibroin, and the artificial fibroin filament is an artificial spider silk fibroin filament. The production of the protein spun yarn according to any one of claims 1 to 3, wherein the crimping step comprises further drying after contacting the artificial fibroin filament or the artificial fibroin staple with the aqueous medium. Method. The method for producing a protein spun yarn according to any one of claims 1 to 4, wherein the aqueous medium used in the crimping step contains a volatile solvent. The method for producing a protein spun yarn according to any one of claims 1 to 5, wherein the artificial fibroin filament contains one type of modified fibroin.

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