JP2022024195A - Fiber-implanted product and fiber-implanting pile - Google Patents

Abstract

To provide a novel fiber-implanted product having a favorable touch feeling.SOLUTION: The fiber-implanted product includes a base material and fibers implanted on the base material, the fibers containing a modified fibroin.SELECTED DRAWING: None

Description

The present invention relates to a flocked product and a pile for flocking.

Products in which piles (short fibers) are attached to the surface of a resin base material via an adhesive are also called flocked products and are used for interior decoration in the housing industry, the automobile industry, and the like. Flocking is carried out for the purposes of heat insulation, sound deadening, prevention of diffused reflection, prevention of dew condensation, friction braking and the like. A flocked product having a good tactile sensation can give further value as a product because it exhibits a heat insulating effect, a sound deadening effect, a good texture, a comfortable feeling, an appearance or a psychologically luxurious feeling.

Further, as a method of flocking piles on the surface of such a base material, an electrostatic flocking method, a fiber coating method and the like are known. In the electrostatic flocking method, when the pile is attached to the surface of the base material coated with the adhesive, a high voltage is applied to the electrodes installed near the base material and above the pile supply container containing the pile. Specifically, a method in which the pile in the lower pile supply container is suspended by electric lines of force and adhered to the surface of the base material, and the pile is dropped toward the surface of the base material so as to be sprayed from above and adhered. The method of making it is known. The fiber coating method is a method in which a pile is sprayed from a spray gun onto a substrate coated with an adhesive to transplant hair (see, for example, Patent Documents 1 and 2). The flocked product obtained by the fiber coating method is flocked with the pile inclined with respect to the surface of the base material.

Japanese Unexamined Patent Publication No. 08-117646 Japanese Unexamined Patent Publication No. 2004-16966

However, when nylon or polyester is mainly used as a flocked pile, it is necessary to increase the basis weight in order to obtain a flocked product with a good tactile sensation, which increases the amount of pile used and reduces the weight of the flocked product. There is a problem that it is difficult.

Therefore, an object of the present invention is to provide a new flocked product having a good tactile sensation. It is also an object of the present invention to provide a pile for flocking.

The present invention provides the following [1] to [5].
[1] A flocked product comprising a base material and fibers planted on the base material, wherein the fibers contain modified fibroin.
[2] The flocked product according to [1], which has a basis weight of 150 g / m ² or less.
[3] A method for producing a flocked product, comprising a step of applying an adhesive to the surface of a base material and a step of implanting fibers containing modified fibroin on the surface of the base material to which the adhesive has been applied.
[4] The method for producing a flocked product according to [3], wherein the basis weight is 150 g / m ² or less.
[5] A pile for flocking containing modified fibroin.

According to the present invention, it is possible to provide a flocked product having a good tactile sensation. In particular, the flocked product according to the present invention exhibits a good tactile sensation even when the basis weight is small.

It is a schematic diagram which shows the domain sequence of the modified fibroin which concerns on one Embodiment. It is a figure which shows the distribution of the value of z / w (%) of the fibroin of natural origin. It is a figure which shows the distribution of the value of x / y (%) of the fibroin of natural origin. It is a schematic diagram which shows the domain sequence of the modified fibroin which concerns on one Embodiment. It is a schematic diagram which shows the domain sequence of the modified fibroin which concerns on one Embodiment.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. It should be noted that the drawings are partially exaggerated for ease of understanding, and the dimensional ratios and the like are not limited to those described in the drawings.

The flocked product according to the embodiment of the present invention can be obtained by flocking a fiber containing modified fibroin on a substrate.

<Base material>
The base material may be any tangible material having a surface (region to be flocked) to which an adhesive for flocking can be applied, and can be appropriately selected depending on the use of the flocked product. The region to be flocked may be a flat surface or a curved surface such as a spherical surface. The area to be flocked may be the entire surface or a part of the substrate.

The material of the base material is a thermoplastic resin such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyester, polyamide, ABS resin (acrylonitrile, butadiene, styrene copolymer synthetic resin); phenol resin, urea resin, melamine resin, epoxy resin. Thermosetting resin such as; may be metal, rubber or ceramic. Further, the base material may be a fiber, a woven fabric made from the fiber, a non-woven fabric, or a knitted fabric.

<Pile for flocking>
The flocking pile (staples) contains the modified fibroin described below. In addition to the modified fibroin, the flocking pile may further contain a pile generally known in the art. Examples of such piles include piles made of nylon and polyester.

The length of the pile may be 0.2 to 1 mm or 1 to 10 mm. When the length of the pile is within the above range, it is easy to show appropriate smoothness, suppleness, and flexibility after flocking, and it is possible to feel stiffness (resistance) and swelling, and the tactile sensation can be improved.

The fineness of the pile may be 0.5 to 2 decitex or 2 to 4 decitex. When the fineness of the pile is in the above range, it is easy to show appropriate smoothness, suppleness, and flexibility after flocking, and the tactile sensation can be improved.

<Modified fibroin>
The modified fibroin according to the present embodiment has a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein contained. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.

As used herein, the term "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be fibroin having the same amino acid sequence as that of naturally occurring fibroin. "Naturally derived fibroin" as used herein is also represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a domain sequence to be used.

The "modified fibroin" may be one in which the amino acid sequence of naturally-derived fibroin is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived). It may be an amino acid sequence modified by modifying the gene sequence of fibroin, or an artificially designed and synthesized one (for example, a nucleic acid encoding the designed amino acid sequence) regardless of naturally occurring fibroin. It may have a desired amino acid sequence by chemical synthesis).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to (A) _n motif of an amino acid sequence) and an amorphous region (typically to REP of an amino acid sequence). It is an amino acid sequence that produces (corresponding to)), and is an amino acid represented by the formula 1: [(A) _n motif-REP] _m or the 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 the number of amino acid residues is 2 to 27. (A) The number of amino acid residues of the _n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (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). A plurality of (A) _n motifs present in a domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m indicates an integer of 2 to 300, and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

The modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. It can be obtained by modifying. Substitutions, deletions, insertions and / or additions of amino acid residues can be carried out by methods well known to those skilled in the art, such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Enzymeology, 100, 448 (1983).

Naturally-derived fibroin is a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Yes, specifically, for example, fibroin produced by insects or spiders.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Anteraea yamamai, Anteraea perni, and tussah. ), Silk moth (Samia synthia), Chrysanthemum (Caligra japonica), Tussar moth (Antheraea mylitta), Muga silk moth (Antheraea assama), silk moth (Visa) Hornet silk protein can be mentioned.

More specific examples of insect-produced fibroin include, for example, the silk moth fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).

Examples of the fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne) such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, etc. Spiders belonging to (Gasteracantha genus), spiders belonging to the spider genus Ordgarius such as Mameitaiseki spider and Mutsutogei sekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc. Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, and spiders belonging to the genus Cytophora. ) Spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Tetragnatha, such as the spider Yasagata spider, Harabiroashidaka spider, and Urokoa spider, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as spiders, spiders belonging to the genus Dyschiriognatha such as spiders, spiders such as spiders, spiders, spiders Spiders belonging to the genus (Latrodectus) and spiders belonging to the spider family (Tetragnatidae family) such as spiders belonging to the genus Euprostenops (Euprosthenops genus) produce spiders. Examples include pider silk protein. Examples of the spider silk protein include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2) and the like.

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (nucleotide sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank sequence AAC47011) ), U37520 (base sequence)), major amplify protein 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (nucleotide sequence), EF595246 (base sequence)), dragline silk protein-derived from nuclear fibroin 2 Numbers AAL32472 (nucleic acid sequence), AF441245 (nucleic acid sequence)), major aminos protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (nucleotide sequence), AJ973155 (nucleotide sequence)), and major protein (GenBank accession number CAM32249.1 (nucleic acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila clavipes] (GenBank accession number AAC145891 (amino acid sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1. The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

As a more specific example of naturally derived fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidoline, amplify, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

The modified fibroin according to the present embodiment may be modified silk fibroin (modified amino acid sequence of silk protein produced by silkworm), or modified spider silk fibroin (spider silk protein produced by spiders). It may be a modified amino acid sequence). As the modified fibroin, modified spider silk fibroin is preferable.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large spitting tube bookmark thread protein produced in the large bottle-shaped gland of spider, and modified fibroin with a reduced content of glycine residues. (Second modified fibroin), (A) Modified fibroin with reduced _n -motif content (third modified fibroin), glycine residue content, and (A) _n -motif content reduced. It has a modified fibroin (fourth modified fibroin), a modified fibroin having a domain sequence containing a region having a locally high hydrophobicity index (fifth modified fibroin), and a domain sequence having a reduced content of glutamine residues. Modified fibroin (sixth modified fibroin) can be mentioned.

The first modified fibroin includes a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, (A) the number of amino acid residues of the _n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, and an integer of 10 to 20. Is even more preferable, an integer of 4 to 16 is even more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the first modified fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. Is even more preferable, and 20 to 75 residues are even more preferable. In the first modified fibroin, the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m is an amino acid residue. It is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number.

The first modified fibroin contains the unit of the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m , and the C-terminal sequence is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 or It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C end of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C end of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.

As a more specific example of the first modified fibroin, (1-i) the amino acid sequence shown by SEQ ID NO: 4 (recombinant spider silk protein ADF3 KaiLageNRSH1), or (1-ii) the amino acid sequence shown by SEQ ID NO: 4 and 90. % And above, modified fibroin containing an amino acid sequence having sequence identity can be mentioned. The sequence identity is preferably 95% or more.

The amino acid sequence represented by SEQ ID NO: 4 is the first to the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 5) consisting of a starting codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in SEQ ID NO: 3.

The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 4.

The second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with naturally occurring fibroin. ..

The second modified fibroin has a domain sequence of GGX and GPGXX in REP as compared with naturally occurring fibroin (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine. In at least one motif sequence selected from), one having an amino acid sequence corresponding to one or more glycine residues in the motif sequence being replaced with another amino acid residue. May be.

In the second modified fibroin, 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 with respect to the total motif sequence.

The second modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and is located closest to the C-terminal side of the domain sequence (A) from the _n motif to the domain sequence. The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence. Therefore, when the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the above domain sequence is w, z / w is 30% or more. It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable. 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 described below.

The method of calculating z / w will be described in more detail. First, in a fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , it is located most on the C-terminal side from the domain sequence (A) _n . The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C end 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. Further, for example, when X (center X) contained in two XGXs exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). be). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A) _n motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

Here, z / w in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Naturally derived fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m among all the extracted fibroins, and the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less. From the amino acid sequence of fibroin in No. 1, z / w was calculated by the above-mentioned calculation method. The results are shown in FIG. The horizontal axis of FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, z / w in naturally-derived fibroin is less than 50.9% (the highest is 50.86%).

In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. Is even more preferable, and 80% or more is even more preferable. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

The second modified fibroin is, for example, modified from the cloned naturally occurring fibroin gene sequence by substituting at least a part of the base sequence encoding the glycine residue to encode another amino acid residue. Obtainable. 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. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment 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 modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be performed.

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

As a more specific example of the second modified fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT468) or SEQ ID NO: 9 (Met). -Contains an amino acid sequence set forth in PRT799) or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. A modified fibroin can be mentioned.

The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. In the amino acid sequence shown in SEQ ID NO: 7, every two (A) _n motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence is further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a part of glutamine (Q) residue is further added. It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have almost the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

The value of z / w in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 9 are 58.7%, respectively. It is 70.1%, 66.1% and 70.0%. Further, the values of x / y in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are They are 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.

The modified fibroin of (2-i) may consist of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of) is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w. Is preferably 50.9% or more.

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

As the tag sequence, for example, an affinity tag using specific affinity (binding, affinity) with other molecules can be mentioned. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chromatography) is performed. Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).

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

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

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. By treating the protein adsorbed via the tag sequence with protease, the modified fibroin from which the tag sequence has been separated can also be recovered.

As a more specific example of the modified fibroin containing the tag sequence, the amino acid represented by (2-iii) SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT468) or SEQ ID NO: 15 (PRT799). Examples may be a modified fibroin comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (2-iv) SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

The amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown by SEQ ID NO: 11 (including His tag sequence and hinge sequence) is added to the N-terminal of the indicated amino acid sequence.

The modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of) is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w. Is preferably 50.9% or more.

The second modified fibroin may contain a secretory 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.

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

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

The third modified fibroin has a domain sequence of at least 1 to 3 (A) _n motifs for every 1 to 3 (A) _n motifs from the N-terminal side to the C-terminal side as compared with naturally occurring fibroin. May have an amino acid sequence corresponding to the deletion of.

The third modified fibroin has a domain sequence of at least two consecutive (A) _n -motif deletions and one (A) from the N-terminal side to the C-terminal side as compared to naturally occurring fibroin. ) It may have an amino acid sequence corresponding to the deletion of the _n -motif being repeated in this order.

The third modified fibroin may have an amino acid sequence corresponding to the deletion of the (A) _n motif at least every other two domain sequences from the N-terminal side to the C-terminal side. ..

The third modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -When the number of amino acid residues in the REP of the unit is sequentially compared and the number of amino acid residues in the REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 1. When x is the maximum value of the sum of the amino acid residues of two adjacent [(A) _n -motif-REP] units, which is 11.3, and y is the total number of amino acid residues of the domain sequence. In addition, 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. (A) The number of alanine residues with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _n motif-first REP (50 amino acid residue)-(A) _n motif-second REP (100 amino acid residue)-(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 an arrangement called _n motifs.

Two adjacent [(A) _n -motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, there may be a [(A) _n motif-REP] unit that is not selected. In FIG. 1, pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. There are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two selected adjacent [(A) _n motif-REP] units is compared. The comparison is made by finding the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is set to 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparison between the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), the 5th REP is assumed to be 1 when the 4th REP having a smaller number of amino acid residues is 1. The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is 1.8 to _11.3 . Shown by a solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than _11.3 . Indicated.

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

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

In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% 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 jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which at least 7 of (A) _n motifs present in a plurality of (A) n motifs are composed only of alanine residues, x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is even more preferable, 70% or more is even more preferable, and 80% or more is used. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

Here, x / y in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Of all the extracted fibroins, x / y from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _m by the above calculation method. Was calculated. The results when the jagged ratio is 1: 1.9 to 4.1 are shown in FIG.

The horizontal axis of FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is clear from FIG. 3, x / y in naturally occurring fibroin is less than 64.2% (highest, 64.14%).

The third modified fibroin, for example, deletes one or more of the sequences encoding the (A) _n motif from the cloned naturally occurring fibroin gene sequence so that x / y is 64.2% or more. Can be obtained by Further, for example, an amino acid sequence corresponding to the deletion of one or more (A) _n motifs so that x / y is 64.2% or more is designed and designed from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing the nucleic acid encoding the amino acid sequence. In each case, in addition to the modification corresponding to the deletion of (A) _n motif from the amino acid sequence of naturally occurring fibroin, further substitution, deletion, insertion and / or addition of one or more amino acid residues. The amino acid sequence corresponding to the above may be modified.

As a more specific example of the third modified fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT468) or SEQ ID NO: 9 (Met). -Contains an amino acid sequence set forth in PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. A modified fibroin can be mentioned.

The modified fibroin of (3-i) will be described. The amino acid sequence shown by SEQ ID NO: 17 is every two (A) _n from the N-terminal side to the C-terminal side from the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. The motif is deleted, and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.

The value of x / y in the Giza ratio 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 15.0%. The value of x / y in the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.3%. The values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. 1% and 70.0%.

The modified fibroin of (3-i) may consist of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by the 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 represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has N-terminal to C-terminal sides. When the number of amino acid residues of REP of two adjacent [(A) _n -motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other The amino acid residue of two adjacent [(A) _n -motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3). When x is the maximum value of the total value obtained by adding the radix and the total number of amino acid residues in the domain sequence is y, it is preferable that x / y is 64.2% or more.

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

As a more specific example of the modified fibroin containing the tag sequence, the amino acid represented by (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT468) or SEQ ID NO: 15 (PRT799). Examples may be a modified fibroin comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (3-iv) SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 11 at the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown by (including His tag sequence and hinge sequence) is added.

The modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n -motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other The maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) _n -motif-REP] units in which the ratio of the number of amino acid residues of REP is 1.8 to 11.3 is x. , When the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

The third modified fibroin may contain a secretory 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.

The fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motifs and a reduced content of glycine residues as compared with naturally occurring fibroin. It has. The domain sequence of the fourth modified fibroin lacks at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with another amino acid residue. That is, it is a modified fibroin having the characteristics of the above-mentioned second modified fibroin and the third modified fibroin. Specific embodiments and the like are as described in the second modified fibroin and the third modified fibroin.

As more specific examples of the fourth modified fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT468), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410). ), The amino acid sequence represented by SEQ ID NO: 14 (PRT468) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. Can be mentioned as a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in. Specific embodiments of the modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

The fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a high hydrophobicity index as compared with naturally occurring fibroin, and / or REP. It may have an amino acid sequence containing a region having a large hydrophobic index locally, which corresponds to the insertion of one or a plurality of amino acid residues having a large hydrophobic index.

It is preferable that the region having a large local hydrophobicity index is composed of consecutive 2 to 4 amino acid residues.

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

In the fifth modified fibroin, one or more amino acid residues in REP were replaced with amino acid residues having a large hydrophobicity index as compared with naturally occurring fibroin, and / or one or more amino acid residues in REP. In addition to the modification corresponding to the insertion of an amino acid residue with a high hydrophobicity index, one or more amino acid residues were substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be a modification of the amino acid sequence corresponding to the above.

The fifth modified fibroin retains hydrophobic amino acid residues, for example, one or more hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydrophobicity index) from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting for a group (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in the REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the 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 either case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acids in the REP. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and extends from the (A) _n motif located most on the C-terminal side to the C-terminal of the above domain sequence. In all REPs contained in the sequence excluding the sequence from the above domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more is defined as p. When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, p / q is 6 It may have an amino acid sequence of .2% or more.

For the hydrophobicity index of the amino acid residue, a known index (Hydrotherapy index: Kyte J, & Dollittle R (1982) “A simple method for dispensing the hydropathic character of B. 105-132) is used. Specifically, the hydrophobicity index of each amino acid (hydropathy index, hereinafter also referred to as “HI”) is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m . (Hereinafter referred to as "array A") is used. First, the average value of the hydrophobicity index of consecutive four amino acid residues is calculated for all REPs contained in the sequence A. The average value of the hydrophobicity index is obtained by dividing the total 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 hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive 4 amino acid residues having an average value of 2.6 or more of hydrophobicity index", it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in the sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average value of hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of 6.6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are present in an overlapping manner by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues is present. A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 4, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) _n motif present at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (eg, a hydrophobic index) in the REP so that the amino acid sequence of the cloned naturally derived fibroin satisfies the above p / q condition. (Amino acid residue with a negative value) is replaced with a hydrophobic amino acid residue (eg, an amino acid residue with a positive hydrophobicity index), and / or one or more hydrophobic amino acid residues are inserted in the REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. It can also be obtained, for example, by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more amino acid residues in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring fibroin, and / or one or more in the REP. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. ..

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ), More preferably valine (V), leucine (L) and isoleucine (I).

As a more specific example of the fifth modified fibroin, (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), Alternatively, modified fibroin comprising (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can be mentioned.

The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 19 is such that the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) is inserted into two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP. A part of the amino acid on the C-terminal side is deleted so as to have substantially the same molecular weight as the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence shown by SEQ ID NO: 20 is the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT468) in which one amino acid sequence (VLI) consisting of 3 amino acid residues is inserted every other REP. be. The amino acid sequence shown in SEQ ID NO: 21 is the amino acid sequence shown in SEQ ID NO: 8 with two amino acid sequences (VLI) consisting of three amino acid residues inserted every other REP.

The modified fibroin of (5-i) may consist of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin of (5-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by the 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 represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most on the C-terminal side (A) _n . Amino acids contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p, and the total number of amino acid residues contained in the sequence excluding the sequence from the _n -motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

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

As a more specific example of the modified fibroin comprising a tag sequence, the amino acid sequence set forth in (5-iii) SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv). ) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

The amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including array and hinge array) is added.

The modified fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

The modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by the 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 represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located most on the C-terminal side (A) _n . Amino acids contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p, and the total number of amino acid residues contained in the sequence excluding the sequence from the _n -motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

The fifth modified fibroin may contain a secretory 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.

The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring fibroin.

The sixth modified fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

When the sixth modified fibroin contains the GPGXX motif in the REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is 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.

In the present specification, 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) Fibroin containing a domain sequence represented by _n -motif (modified fibroin or naturally derived). In (fibroin), the number of GPGXX motifs contained in the region in all REPs included in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. Let s be the number obtained by multiplying the total number by 3 (that is, corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence. The GPGXX motif content 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, the target of "the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is "the most C-terminal side". (A) Sequence from _n motif to C end of domain sequence "(sequence corresponding to REP) may contain a sequence characteristic of fibroin and a sequence having low correlation, and m is small. This is because the case (that is, when the domain sequence is short) affects the calculation result of the GPGXX motif content, and this effect is eliminated. When the "GPGXX motif" is located at the C-terminal of the REP, even if the "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 5 is a schematic diagram showing the domain sequence of modified fibroin. The calculation method of the GPGXX motif content will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 5 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “located closest to the C-terminal side”. (A) Since it is included in the "sequence obtained by removing the sequence from the _n motif to the C-terminal of the domain sequence from the domain sequence" (the sequence shown by "region A" in FIG. 5), s is calculated. 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 most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (the sequence shown in "Region A" in FIG. 5). Since it is contained in.), The total number t of amino acid residues in all REPs 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% in the case of the modified fibroin of FIG.

The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, further preferably 4% or less, and particularly preferably 0%. ..

In the present specification, the "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) Fibroin containing a domain sequence represented by the _n -motif (modified fibroin or naturally derived). In fibroin), all the sequences contained in the sequence excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In REP, the total number of glutamine residues contained in the region is u, and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal 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 the motif. In the calculation of the glutamine residue content, the reason for targeting "the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin corresponds to its domain sequence being deleted from one or more glutamine residues in REP or replaced with other amino acid residues as compared to naturally occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and 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 preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are more preferably amino acid residues.

The sixth modified fibroin preferably has a hydrophobicity of REP of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present specification, 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) Fibroin containing a domain sequence represented by the _n -motif (modified fibroin or naturally derived). In fibroin), all the sequences contained in the sequence excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In REP, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal 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 of all REPs excluding the _n motif. In the calculation of the hydrophobicity of REP, the reason for targeting "the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in REP. In addition to the modification corresponding to the substitution with another amino acid residue, there may be further modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. ..

The sixth modified fibroin is, for example, deleted one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and / or other than one or more glutamine residues in REP. It can be obtained by substituting with the amino acid residue of. Also, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As more specific examples of the sixth modified fibroin, (6-i) SEQ ID NO: 25 (M_PRT888), SEQ ID NO: 26 (M_PRT965), SEQ ID NO: 27 (M_PRT888), SEQ ID NO: 28 (M_PRT916), SEQ ID NO: 29 ( M_PRT918), modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 30 (M_PRT699), SEQ ID NO: 31 (M_PRT698) or SEQ ID NO: 32 (Met-PRT966), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32.

The modified fibroin of (6-i) will be described. The amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting VL for all the QQs in the amino acid sequence (Met-PRT410) shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 26 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with TS, and the remaining Qs are replaced with A. In the amino acid sequence shown by SEQ ID NO: 27, all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VL, and the remaining Qs are replaced with I. The amino acid sequence shown in SEQ ID NO: 28 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VI, and the remaining Qs are replaced with L. The amino acid sequence shown in SEQ ID NO: 29 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VF, and the remaining Qs are replaced with I.

The amino acid sequence shown in SEQ ID NO: 30 is obtained by substituting VL for all the QQs in the amino acid sequence (Met-PRT468) shown in SEQ ID NO: 8. In the amino acid sequence shown by SEQ ID NO: 31, all QQs in the amino acid sequence shown by SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.

In the amino acid sequence shown in SEQ ID NO: 32, all the QQs in the sequence in which the regions of the 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) are repeated twice are replaced with VF. And the remaining Q is replaced with I.

The amino acid sequences represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 all have a glutamine residue content of 9% or less. There is (Table 2).

The modified fibroin of (6-i) comprises the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. There may be.

The modified fibroin of (6-ii) is 90% or more of the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. It contains an amino acid sequence having the sequence identity of. The modified fibroin of (6-ii) is also a domain represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

As more specific examples of the modified fibroin containing the tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37. (PRT918), modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698) or SEQ ID NO: 40 (PRT966), or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: Examples include modified fibroins comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.

The amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 are SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively. , SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 added to the N-terminal of the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence). It is a thing. Since only the tag sequence was added to the N-terminal, there was no change in the glutamine residue content, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39. And the amino acid sequence shown by SEQ ID NO: 40 has a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) comprises the amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. There may be.

The modified fibroin of (6-iv) is 90% or more of the amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. It contains an amino acid sequence having the sequence identity of. The modified fibroin of (6-iv) is also a domain represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

The sixth modified fibroin may contain a secretory 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.

The modified fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. It may be a modified fibroin having the above-mentioned characteristics.

<Manufacturing method of modified fibroin>
The modified fibroin according to the present embodiment is, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing the nucleic acid.

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) using a gene encoding natural fibroin and modifying it by a genetic engineering method, or a method of chemically synthesizing it. be able to. The chemical synthesis method of nucleic acid is not particularly limited, and for example, AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.) based on the amino acid sequence information of fibroin obtained from the NCBI web database or the like. Genes 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 modified fibroin, a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a starting codon and a His10 tag is added to the N-terminal of the above amino acid sequence is synthesized. You may.

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

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector that can be autonomously replicated in a host cell or can be integrated into the chromosome of the host and contains a promoter at a position where a nucleic acid encoding modified fibroin can be transcribed is preferably used.

As the host, any of 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, Brevibacillus, Corinebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satirus and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Phyllum and the like. Examples of the microorganism belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, the vector into which the nucleic acid encoding the modified fibroin is introduced includes, for example, pBTrp2 (manufactured by Behringer-Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, Examples thereof include pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569).

Examples of eukaryotic hosts include yeast and filamentous fungi (molds and the like). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of the filamentous fungus include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

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

As a method for expressing nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. ..

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

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and the host can be efficiently cultured. If so, either a natural medium or a synthetic medium may be used.

The carbon source may be any assimilated by the transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolysates, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used. Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used. As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted by using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

Further, during the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed. 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 needed. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

The expressed modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer solution, and then an ultrasonic crusher, a French press, or a manton. Crush the host cells with a gaulin homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, the methods usually used for isolating and purifying modified fibroin, that is, solvent extraction method, salting-out method using ammonium sulfate, desalting method, organic solvent, etc. Anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -cepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), and a resin such as S-Sepharose FF (manufactured by Pharmacia). Cation exchange chromatography method, hydrophobic chromatography method using resin such as butyl Sepharose, phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis such as isoelectric point electrophoresis, etc. Purified preparations can be obtained by using methods such as the law alone or in combination.

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble material of the modified fibroin is recovered as a precipitate fraction by similarly collecting the host cells, disrupting the cells, and centrifuging the cells. The insoluble material of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified specimen of modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant and using the same isolation and purification method as described above from the culture supernatant.

Another embodiment of the present invention is a method for producing a flocked product. The method includes a step of applying an adhesive to the surface of the base material (coating step) and a step of flocking fibers containing modified fibroin on the surface of the base material to which the adhesive is applied (flocking step). ..

The adhesive may be any generally known in the art, and may be, for example, a rubber-based adhesive, an epoxy-based adhesive, an acrylic-based adhesive, a urethane-based adhesive, or a silicon-based adhesive. May be good. Further, the adhesive may be a moisture-curable one-component adhesive, a two-component mixed adhesive, an emulsion-type adhesive, or a hot-melt type adhesive.

In the coating step, an adhesive layer is formed on the surface of the base material by applying the adhesive. Generally, an adhesive layer is formed over the entire area to be flocked. In the coating step, an adhesiveness improving agent may be used before applying the adhesive.

The flocking step is a step of adhering the pile to the adhesive layer formed in the coating step by a flocking method known to those skilled in the art. The flocking method may be a method generally known in the art, and may be an electrostatic flocking method or a fiber coating method.

The basis weight of the flocked product is not particularly limited. Even if the grain size is 50 g / m ² or more, 60 g / m ² or more, 70 g / m ² or more, 80 g / m ² or more, 90 g / m ² or more, 95 g / m ² or more, or 100 g / m ² or more. It may be 300 g / m ² or less, 270 g / m ² or less, 240 g / m ² or less, 210 g / m ² or less, 180 g / m ² or less, or 150 g / m ² or less. With the flocked product according to the present embodiment, even if the basis weight is 150 g / m ² or less, the weight of the flocked product can be reduced without deteriorating the tactile sensation.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.

[Manufacturing of modified fibroin]
(1) Construction of expression vector A modified fibroin (PRT799) having the amino acid sequence shown by SEQ ID NO: 15 and a modified fibroin (PRT918) having the amino acid sequence shown by SEQ ID NO: 37 were designed.

Nucleic acids encoding proteins with the designed amino acid sequences were synthesized respectively. NdeI sites were added to the nucleic acid at the 5'end, and EcoRI sites were added downstream of the stop codon. Each of these two types of nucleic acids was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector.

(2) Expression of modified fibroin Escherichia coli BLR (DE3) was transformed with the obtained expression vectors. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture broth was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that OD ₆₀₀ was 0.005. The culture solution temperature was maintained at 30 ° C., and flask culture was carried out until the OD ₆₀₀ reached 5, (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD ₆₀₀ was 0.05, and transformed Escherichia coli was inoculated. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. In addition, 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 culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the desired modified fibroin. Twenty hours after the addition of IPTG, the culture broth was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture medium before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of the band of the desired modified fibroin size depending on the addition of IPTG. did.

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

[Manufacturing of raw yarn (molded body)]
(1) Preparation of Spinning Solution (Dope Solution) Using DMSO in which lithium chloride was dissolved so as to be 4% by mass as a solvent, the freeze-dried powder of the modified fibroin (PRT799 or PRT918) produced above was used at a concentration of 24% by mass. It was added to the solvent so as to be. After melting with an aluminum block heater at 90 ° C. for 1 hour, insoluble matter and bubbles were removed to obtain a spinning liquid (doping liquid).

(2) Spinning A reserve tank was filled with a spinning liquid and discharged from a 0.1 or 0.2 mm diameter monohole nozzle into a 100% by mass methanol coagulation bath using a gear pump. The discharge rate was adjusted to 0.01 to 0.08 mL / min. After solidification, washing and stretching were performed in a 100% by mass methanol washing bath. After washing and stretching, it was dried using a dry heat plate, and the obtained raw yarn (modified fibroin fiber) was wound up.

[Manufacturing of flocked products]
(Comparative Example 1)
A polypropylene flat plate (100 mm × 100 mm × 2 mm) was prepared as a base material for flocking. As a pile for flocking, a nylon pile (length: 1 mm, thickness: 3D) manufactured by Chubu Pile Industry Co., Ltd. was prepared. A water-soluble emulsion type adhesive is applied to the surface of the base material, and the nylon pile is applied with an electrostatic flocking machine (trade name: CP70, manufactured by Eishin Air Conditioning Co., Ltd.) with a grain size of approximately 200 g / m. The flocked product of Comparative Example 1 was obtained by electrostatically flocking so as to be ² .

(Comparative Example 2)
A hair transplanted product of Comparative Example 2 was obtained in the same manner as in Comparative Example 1 except that the hair was electrostatically transplanted so that the basis weight was about 150 g / m ² .

(Example 1)
A polypropylene flat plate (100 mm × 100 mm × 2 mm) was prepared as a base material for flocking. Staples (length: 1 mm, thickness: 1.3D) containing modified fibroin PRT799 were prepared as a pile for flocking.
A water-soluble emulsion type adhesive (trade name: SB22K, manufactured by Henkel Japan Ltd.) is applied to the surface of the above substrate, and an electrostatic flocking machine (trade name: CP70, manufactured by Eishin Air Conditioning Co., Ltd.) is used. The short fibers were electrostatically transplanted so as to have a grain size of about 150 g / m ² , and the hair transplanted product of Example 1 was obtained.

(Example 2)
A polypropylene flat plate (100 mm × 100 mm × 2 mm) was prepared as a base material for flocking. Staples (length: 1 mm, thickness: 1.3D) containing modified fibroin PRT918 were prepared as a pile for flocking.
A water-soluble emulsion type adhesive is applied to the surface of the base material, and the staple fibers are coated with an electrostatic flocking machine (trade name: CP70, manufactured by Eishin Air Conditioning Co., Ltd.) at a grain size of about 100 g / m. The hair-transplanted product of Example 2 was obtained by electrostatically transplanting hair so as to be ² .

Table 6 shows the basis weight of each of the obtained flocked products. The basis weight was calculated based on the difference between the weight of the flocked product and the weight of the base material alone.

[Evaluation of tactile sensation]
Since there is no universal evaluation method for tactile sensation, the materials used for automobile interior parts, etc., which are arranged in the order in which they are recognized as having good tactile sensation in the industry, are evaluated as an index.
Specifically, each of the 100 subjects corresponds to which score the tactile sensation when touching the flocked product of Example 1 or 2 after touching the material corresponding to the scores 1 to 7 shown in Table 7. Was evaluated.
In Table 7, Ultrasuede (trade name, Toray Industries, Inc.) is a composite fiber of ultrafine raw yarn of polyester and polyurethane having a homogeneous sea-island structure, and is a polymer elastic body with only nodal points between the fibers. It is a non-woven fabric that also retains a fixed microporous structure.

The scores of Examples 1 and 2 were both 4. In particular, considering the tendency that the larger the basis weight amount is, the better the feel of touch is, the basis weight of Examples 1 and 2 is smaller than that of Comparative Example 1, but the same feel as that of Comparative Example 1. showed that.

[Evaluation of adhesiveness]
The adhesiveness of each flocked product was evaluated according to the following procedure.
(1) In the same manner as in Examples 1 and 2 and Comparative Examples 1 and 2, 3 g of each flocking pile is electrostatically flocked on the substrate.
(2) Each of the obtained flocked products is placed on a flat surface (the size of one square is 10 mm × 10 mm) having a grid-like dividing line at 10 mm intervals, and the flocked surface faces downward. Fix it in the air like this.
(3) A weight of 500 g is dropped from a height of 50 mm above the back surface of the flocked product (the surface opposite to the flocked surface) to collide with the flocked product.
(4) Visually count the number of fallen piles (using a magnifying glass if necessary) for each square. Six squares are selected in descending order of the number of fallen piles, and the average value of the number of fallen piles per square is calculated.

The results are shown in Table 8. In Comparative Examples 1 and 2, 20 or more piles fell per square, whereas in Examples 1 and 2, less than 4 piles fell per square. Examples 1 and 2 in which short fibers containing modified fibroin were transplanted had significantly improved adhesiveness as compared with Comparative Examples 1 and 2 in which nylon pile was transplanted.

Claims (3)

A base material and fibers planted on the base material are provided.
A flocked product in which the fiber contains modified fibroin. The flocked product according to claim 1, wherein the basis weight is 150 g / m ² or less. A pile for flocking containing modified fibroin.

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