JP2021054771A - Water retention-imparting agent and method of imparting water retention - Google Patents

Abstract

To provide a water retention-imparting agent that can easily impart water retention to various materials or articles in a simple process, and to provide a method that can easily impart water retention to a given material or article.SOLUTION: A water retention-imparting agent comprises modified spider silk fibroin as an active ingredient. A method for imparting water retention to an article comprises a step of incorporating modified spider silk fibroin into the article.SELECTED DRAWING: None

Description

The present invention relates to a water retention imparting agent and a method for imparting water retention.

Products and raw materials that require water retention are required in various fields. Various proposals have been made so far for technical means for obtaining products (for example, fibers) having improved functions such as water retention. For example, with respect to polyamide fibers, a core-sheath type composite fiber containing an alkali-soluble polyester in a core and a sheath is formed by using a composite spinning device to elute the alkali-soluble polyester, and each single yarn has a hollow portion and a large number. It is disclosed that a fine-pore hollow polyamide fiber is obtained by generating the fine pores of the above (Patent Document 1).

Further, for example, regarding protein fibers, a cocoon is formed using a recombinant silk moth in which a nucleic acid encoding the water-soluble protein is introduced into the silk moth so as to generate a water-soluble protein in the posterior silk gland of the silk moth, and fibroin is formed from the cocoon. A method for obtaining a protein fiber by obtaining a silk fiber having a water-soluble protein portion existing inside the portion is disclosed (Patent Document 2).

JP-A-2007-332486 Japanese Unexamined Patent Publication No. 2014-198715

However, in the method disclosed in the above document, it is unavoidable that the manufacturing process of various materials or articles becomes complicated in order to improve the water retention.

The present invention provides a water-retaining agent capable of imparting water retention to various materials or articles in a simple process, and a method capable of easily imparting water retention to various articles. The purpose.

The present inventors have found that the modified spider silk fibroin has excellent water retention. The present invention is based on this novel finding.

The present invention relates to, for example, the following inventions.
[1]
A water-retaining agent containing modified spider silk fibroin as an active ingredient.
[2]
The water-retaining agent according to [1], wherein the modified spider silk fibroin contains the modified spider silk fibroin.
[3]
The water-retaining agent according to [1] or [2], wherein the modified spider silk fibroin contains a modified spider silk fibroin having an average hydrophobicity index of −1.0 or more.
[4]
The water-retaining agent according to any one of [1] to [3], which is in the form of powder, liquid or fiber.
[5]
The water-retaining agent according to any one of [1] to [4], which is in the form of fibers.
[6]
A method of imparting water retention to an article
A method comprising the step of incorporating modified spider silk fibroin into the article.
[7]
The method according to [6], wherein the modified spider silk fibroin comprises the modified spider silk fibroin.
[8]
The method according to [6] or [7], wherein the modified spider silk fibroin contains a modified spider silk fibroin having an average hydrophobicity index of −1.0 or higher.
[9]
The method according to any one of [6] to [8], which is in the form of powder, liquid or fiber.
[10]
The method according to any one of [6] to [9], which is a form of fiber.

According to the present invention, it is possible to provide a water retention imparting agent capable of imparting water retention to various materials (raw materials) or articles in a simple process. Further, according to the present invention, it is possible to provide a method capable of easily imparting water retention to various articles.

According to the present invention, for example, by mixing a water-retaining agent according to the present invention with a material having biodegradability, water retention can be imparted without impairing the biodegradability. it can. According to the present invention, the degree of water retention of the article can also be adjusted by adjusting the amount of the modified spider silk fibroin (active ingredient) contained in the predetermined article.

It is a schematic diagram which shows an example of the domain sequence of modified spider silk fibroin. It is a figure which shows the distribution of the value of z / w (%) of naturally-derived fibroin. It is a figure which shows the distribution of the value of x / y (%) of naturally-derived fibroin. It is a schematic diagram which shows an example of the domain sequence of modified spider silk fibroin. It is a schematic diagram which shows an example of the domain sequence of modified spider silk fibroin. It is a graph which shows the time-dependent change of the ratio of the water content per fiber weight in an Example.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Water retention agent]
The water-retaining agent according to the present embodiment contains modified spider silk fibroin as an active ingredient. Water retention refers to the property of maintaining a state of retaining water. The water-retaining agent according to the present embodiment utilizes the property of modified spider silk fibroin, which is excellent in water-retaining property.

(Modified spider silk fibroin)
The modified spider silk fibroin according to the present embodiment has 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 modified spider 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 spider silk fibroin" means artificially produced fibroin (artificial spider silk fibroin). The modified spider silk fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of the naturally occurring spider silk fibroin, or may be a fibroin having the same amino acid sequence as the naturally occurring spider silk fibroin. The "naturally-derived spider silk fibroin" as used herein also has 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 represented by.

The "modified spider fibroin" may be one in which the amino acid sequence of the naturally occurring spider fibroin is used as it is, or the amino acid sequence thereof is modified based on the amino acid sequence of the naturally occurring spider fibroin (for example). , Amino acid sequence modified by modifying the gene sequence of cloned naturally occurring spider fibroin), or artificially designed and synthesized regardless of naturally occurring spider fibroin (eg,) , Which has a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically _{corresponding to the (A) n} motif of an amino acid sequence) and an amorphous region (typically to the REP of an amino acid sequence). It is an amino acid sequence that produces (corresponding to.), And is 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, the (A) _n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2-27. (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 the 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, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160, or It may be an amino acid sequence composed of 10 to 180 amino acid residues. m represents an integer of 2 to 300, 8 to 300, 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, It may be an integer of 20 to 140 or 20 to 12010 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.

In the modified spider silk fibroin according to the present embodiment, for example, one or more amino acid residues are substituted, deleted, inserted and / or added to the cloned naturally occurring spider silk fibroin gene sequence. It can be obtained by modifying the corresponding amino acid sequence. Substitution, deletion, insertion and / or addition 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 Energy, 100, 448 (1983).

Naturally-derived spider silk fibroin comprises a domain sequence represented by _{Formula 1: [(A) n} Motif-REP] _m or Formula 2: [(A) _n Motif-REP] _m- (A) _{n Motif.} It is a protein. Specific examples of the naturally occurring arachnid fibroin include fibroin produced by arachnids.

Examples of fibroin produced by arachnids include spider silk proteins produced by spiders belonging to the order Araneae. More specifically, 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, spiders. Spiders belonging to the genus Pronus, such as spiders, spiders belonging to the genus Trinofundamashi, such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, spiders, spiders, spiders, spiders, etc. Spiders belonging to the genus Ordgarius, 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, spiders Spiders belonging to the genus), spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora such as spiders, spiders, spiders and spiders, and spiders belonging to the genus Poltys such as spiders. Spider silk proteins produced by spiders belonging to the genus Cyclosa, 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 belonging to the genus Tetragnatha, such as Ashidaka spider and Urokoa shinagamo, spiders belonging to the genus White spider (genus Leucage), spiders belonging to the genus Leucage, spiders belonging to the genus Leucage, spiders belonging to the genus Leucage Spiders belonging to the genus Azumi (Menosira genus) such as spiders and spiders, spiders belonging to the genus Dyschiriognatha such as Himea shinagamo, spiders belonging to the genus Dyschiriognatha, spiders, spiders, sea urchins, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprostenops, and spiders belonging to the genus Euprostenops. Be done. 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), AcSp, PySp, Flag 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 numbers AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank sequence No. AAC47011 (amino acid sequence), U47856 (base sequence)) ), U37520 (base sequence)), major amplifier spidroin 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein (derived from radinic protein) Numbers AAL32472 (amino acid sequence), AF441245 (base sequence)), major amplifier protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major protein (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila proteins] (GenBank accession number AAC1458.91 (amino acid sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nefilengys cruisetata] (GenBank access) The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

As a more specific example of the naturally occurring spider silk fibroin, further, spider silk 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, spidroin, complete, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFINITION. 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.

As a specific example of the modified spider fibroin, the content of the modified spider fibroin (first modified spider fibroin) derived from the large spitting tube bookmark thread protein produced in the large bottle-shaped gland of the spider and the glycine residue. Modified spider fibroin having a reduced domain sequence (second modified spider fibroin), (A) Modified spider fibroin having a domain sequence with reduced _{n motif content (third modified spider fibroin)} ), Modified spider fibroin (fourth modified spider fibroin) with reduced glycine residue content, and (A) _n motif content, and a domain sequence containing a region with a locally high hydrophobicity index. Examples thereof include modified spider fibroin (fifth modified spider fibroin) and modified spider fibroin (sixth modified spider fibroin) having a domain sequence having a reduced content of glutamine residues.

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

The first modified spider silk fibroin contains an amino acid sequence unit represented by the formula 1: [(A) _n motif-REP] _m , and the C-terminal sequence is an amino acid represented by any of SEQ ID NOs: 1 to 3. It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of the sequences or 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-terminal of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C-terminal 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 spider silk fibroin, the amino acid sequence shown in (1-i) SEQ ID NO: 4 (recombinant spider silk product ADF3 KaiLargeNRSH1) or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 And modified spider silk fibroin containing an amino acid sequence having 90% or more sequence identity. The sequence identity is preferably 95% or more.

The amino acid sequence shown by SEQ ID NO: 4 is the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 5) consisting of the start codon, the His10 tag and the 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 spider silk fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 4.

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

The second modified spider fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is other than glycine) as compared with naturally occurring spider fibroin. In at least one motif sequence selected from (1), an amino acid sequence corresponding to at least one or a plurality of glycine residues in the motif sequence being replaced with another amino acid residue. It may have.

In the second modified spider silk 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 spider silk fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and is located on the C-terminal side of the above domain sequence from the (A) _n motif described above. Let z be 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 the domain sequence. _{When the total number of amino acid residues in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the above domain sequence from the domain sequence is w, z / w is 30%. As mentioned above, 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 spider silk 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. The content ratio of the amino acid sequence consisting of GGX in the domain sequence of the second modified spider silk fibroin is preferably 30% or less, more preferably 20% or less, still more 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 below.

The method of calculating z / w will be described in more detail. First, in the fibroin (modified spider silk fibroin or naturally-derived spider silk fibroin) containing the 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) _An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the n 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 XGX 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). is there). 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 of arachnid-derived fibroin were extracted) were extracted. Naturally derived fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] m and having an amino acid sequence consisting of GGX in fibroin of 6% or less among all the extracted fibroins. From the amino acid sequence of fibroin, z / w was calculated by the above-mentioned calculation method. The result is shown in FIG. The horizontal axis of FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z / w in the naturally occurring spider silk fibroin is less than 50.9% (the highest is 50.86%).

In the second modified spider silk fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, 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 spider silk fibroin, for example, from the cloned naturally occurring spider silk fibroin gene sequence, replaces at least a part of the nucleotide sequence encoding the glycine residue so as to encode another amino acid residue. It can be obtained by modifying it. 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 replaced 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 aspects from the amino acid sequence of naturally occurring spider fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the substitution of the glycine residue in the REP with another amino acid residue from the amino acid sequence of the naturally occurring spider fibroin, one or more amino acid residues were further substituted. The amino acid sequence corresponding to the deletion, insertion and / or addition may be modified.

The other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than the glycine residue, but is a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and methionine ( Hydrophobic 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) residue and glutamate (E) residue are preferred, valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine ( F) 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 spider silk fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 An amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in (Met-PRT799) or the amino acid sequence shown in (2-ii) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Including, modified spider silk fibroin can be mentioned.

The modified spider silk 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 spider silk fibroin. _{In the amino acid sequence shown by 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 some glutamine (Q) residues are further added.} It was replaced with a serine (S) residue, and some amino acids on the C-terminal side were deleted so as to have substantially 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). A predetermined hinge sequence and His tag sequence are 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 spider silk 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 value of x / y in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is They are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.

The modified spider silk 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 spider 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 spider silk 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 spider 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. When the total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) is z and the total number of amino acid residues in REP in the domain sequence is w, z / W is preferably 50.9% or more.

The second modified spider silk 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 spider silk fibroin.

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. 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. It can be used for isolation. 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 spider silk 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 a protease, the modified spider silk fibroin from which the tag sequence has been separated can also be recovered.

More specific examples of the modified spider silk fibroin containing the tag sequence are shown in (2-iii) SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). A modified spider silk fibroin comprising the amino acid sequence (2-iv), or 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. Can be mentioned.

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 spider silk 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 spider 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 spider silk 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 spider 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. When the total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) is z and the total number of amino acid residues in REP in the domain sequence is w, z / W is preferably 50.9% or more.

The second modified spider silk 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 spider silk fibroin has an amino acid sequence _{whose domain sequence has a reduced content of (A) n motif as compared with naturally occurring spider silk fibroin.} It can be said that the domain sequence of the third modified spider silk 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 spider silk fibroin.} ..

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

The third modified spider silk fibroin has a domain sequence of at least 1 for every 1 _{to 3 (A) n} motifs from the N-terminal side to the C-terminal side as compared with the naturally occurring spider silk fibroin. A) It may have an amino acid sequence corresponding to the deletion of the _{n motif.}

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

The third modified spider silk fibroin has 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. May be good.

The third modified spider silk fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and two adjacent [(A)) from the N-terminal side to the C-terminal side. _{When the} number of amino acid residues in the REP of the n-motif-REP] 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. Let x be the maximum value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units, which are 8 to 11.3, and let y be the total number of amino acid residues in the domain sequence. It may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (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 spider silk fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n. Motif-Third REP (10 amino acid residues)-(A) _n Motif-Fourth REP (20 amino acid residues)-(A) _n Motif-Fifth REP (30 amino acid residues)-(A) _It has 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 adjacent [(A) n motif-REP] units selected is compared.} The comparison is performed by obtaining 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 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with a smaller number of amino acid residues is set to 1, the fifth REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

In Figure 1, when the 1 more lesser of amino acid residues, the ratio of the other amino acid residues is from 1.8 to 11.3 a set of _{[(A) n} motif -rep] Unit Shown by 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 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 _{. The set of [(A) n} motif-REP] units is indicated by a broken line. 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. is there.). 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 maximized 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 spider silk fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, and more preferably 70% or more. Even more preferably, it is even more preferably 75% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the 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 spider silk fibroin is a modified spider silk fibroin in which _{at least 7 of the (A) n} motifs present in the domain sequence are composed only of alanine residues, x / y is 46.4%. The above is preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, and 70% or more is even more preferable. It is particularly preferably 80% or more. 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 of arachnid-derived fibroin were extracted) were extracted. Of all the extracted fibroin, 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, the x / y of the naturally occurring spider silk fibroin is less than 64.2% (the highest is 64.14%).

The third modified spider silk fibroin is, for example, one or more of the sequences encoding the _{(A) n} motif so that x / y is 64.2% or more from the cloned naturally occurring spider silk fibroin gene sequence. Can be obtained by deleting. Further, for example, from the amino acid sequence of naturally-derived spider silk fibroin, 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.} It can also be obtained by chemically synthesizing a nucleic acid encoding the designed 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 spider fibroin, one or more amino acid residues were further substituted, deleted, inserted and /. Alternatively, the amino acid sequence corresponding to the addition may be modified.

As a more specific example of the third modified spider silk fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 An amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in (Met-PRT799) or the amino acid sequence shown in (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 Including, modified spider silk fibroin can be mentioned.

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

The value of x / y in the jagged ratio of 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 15.0%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 17 and the amino acid sequence shown in 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 89.8%. The values of z / w in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. 1% and 70.0%.

The modified spider silk 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 spider 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 spider silk 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 spider silk 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 is C from the N-terminal side. _{When the number of amino acid residues of REP of two adjacent [(A) n} motif-REP] units is sequentially compared toward the terminal side, and the number of amino acid residues of REP having a small number of amino acid residues is set to 1. , The ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 (the jagged ratio is 1: 1.8 to 11.3) of two adjacent [(A) _n motif-REP] units. When x is the maximum value of the total value obtained by adding the number of amino acid residues and y is the total number of amino acid residues in the domain sequence, x / y is preferably 64.2% or more.

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

More specific examples of the modified spider silk fibroin containing the tag sequence are shown in (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). A modified spider silk fibroin comprising the amino acid sequence (3-iv), or 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. Can be mentioned.

The amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are the N-terminals of the amino acid sequences shown in 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 spider silk 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 spider 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 spider silk 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 spider silk fibroin (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 C from the N-terminal side. _{When the number of amino acid residues of REP of two adjacent [(A) n} motif-REP] units is sequentially compared toward the terminal side, and the number of amino acid residues of REP having a small number of amino acid residues is set to 1. , The maximum value of the total value of the sum of 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 the other REP is 1.8 to 11.3 When x is defined and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

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

As more specific examples of the fourth modified spider silk fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410), the amino acid sequence set forth in SEQ ID NO: 14 (PRT525) 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: Examples thereof include modified spider silk fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by No. 15. Specific embodiments of the modified spider silk 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 spider 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 spider fibroin. And / or it may have an amino acid sequence that locally contains a region with a large hydrophobicity index, which corresponds to the insertion of one or more amino acid residues with a large hydrophobicity index in the REP.

The region having a locally large hydrophobicity index is preferably composed of consecutive 2 to 4 amino acid residues.

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

In the fifth modified spider fibroin, one or more amino acid residues in the REP were replaced with amino acid residues having a higher hydrophobicity index as compared with the naturally occurring spider fibroin, and / or in the REP. In addition to the modification corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index, one or more amino acid residues were substituted or deleted as compared with the naturally occurring spider fibroin. , Insertion and / or addition of amino acid sequences may be modified.

The fifth modified spider fibroin contains, for example, one or more hydrophilic amino acid residues in REP (eg, amino acid residues having a negative hydrophobicity index) from the cloned naturally occurring spider fibroin gene sequence. It can be obtained by substituting a hydrophobic amino acid residue (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 REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acids in REP. It can also be obtained by designing an amino acid sequence corresponding to the insertion of a residue 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 spider silk fibroin, and / or one or more hydrophobic in the REP. In addition to the modification corresponding to the insertion of the sex amino acid 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 spider silk fibroin contains the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and is located closest to the C-terminal side (A) from the _n- motif to the C-terminal of the above domain sequence. In all REPs included in the sequence excluding the sequence up to 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 p. _{When the total number of amino acid residues contained in the sequence obtained by 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 is q, p / q May have an amino acid sequence of 6.2% or more.

For the hydrophobicity index of amino acid residues, a known index (Hydropathic index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic protein, B. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid 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 "sequence A") is used. First, the average value of the hydrophobicity index of four consecutive 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 consecutive 4 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 four amino acid residues having an average value of 2.6 or more of the 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 the 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 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 duplicated by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues 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 the 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 existing 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 spider silk fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and further preferably 20% or more. Is even more preferable, and 30% or more is even more preferable. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

The fifth modified spider silk fibroin comprises, for example, one or more hydrophilic amino acid residues (eg,) in the REP such that the amino acid sequence of the cloned naturally occurring spider silk fibroin is subjected to the above p / q condition. , Amino acid residues with a negative hydrophobicity index) are replaced with hydrophobic amino acid residues (eg, amino acid residues with a positive hydrophobicity index), and / or one or more hydrophobic amino acids in the REP. By inserting a residue, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can be obtained by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally-derived spider silk 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 spider fibroin, and / or one or more in the REP. In addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues is further performed. May be good.

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). ) Is preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.

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

The modified spider silk fibroin of (5-i) will be described. The amino acid sequence shown by SEQ ID NO: 19 consists of 3 amino acid residues every other REP, except for the domain sequence of the terminal on the C-terminal side, with respect to the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410). Two amino acid sequences (VLI) are inserted, a part of glutamine (Q) residue is replaced with a serine (S) residue, and a part of amino acid on the C-terminal side is deleted. The amino acid sequence shown by SEQ ID NO: 20 is the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. is there. 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 spider silk 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 spider silk fibroin of (5-ii) contains 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 spider silk 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 spider silk fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and is located most on the C-terminal side (A). ) In _{all REPs included in the sequence excluding the sequence from the n} motif to the C-terminal of the domain sequence from the domain sequence, it is included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more. Let p be the total number of amino acid residues, and let q be 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 to the C-terminal of the domain sequence from the domain sequence. Occasionally, p / q is preferably 6.2% or higher.

The fifth modified spider silk 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 spider silk 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) Examples thereof include modified spider silk fibroin containing an amino acid sequence having 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.

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

The modified spider silk 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 spider silk 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 spider silk 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 spider silk fibroin (5-iv) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located most on the C-terminal side (A). ) In _{all REPs included in the sequence excluding the sequence from the n} motif to the C-terminal of the domain sequence from the domain sequence, it is included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more. Let p be the total number of amino acid residues, and let q be 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 to the C-terminal of the domain sequence from the domain sequence. Occasionally, p / q is preferably 6.2% or higher.

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

The sixth modified spider silk 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 spider silk fibroin contains the GPGXXX motif in the REP, the content of the GPGXXX motif 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 spider silk fibroin or natural In the derived spider silk fibroin), GPGXX contained in the region in all REPs included 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.} Let s be the number obtained by multiplying the total number of motifs 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 closest to the C-terminal side to the C-terminal of 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 motif.}

In the calculation of the GPGXX motif content, "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 targeted at "the most C-terminal side". (A) The _{sequence from the n} motif to the C-terminal of the domain sequence (the sequence corresponding to REP) may contain a sequence having a low correlation with the sequence characteristic of fibroin, and m is small. In this case (that is, when the domain sequence is short), it 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 "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 5 is a schematic diagram showing the domain sequence of the modified spider silk fibroin. The calculation method of the GPGXX motif content rate will be specifically described with reference to FIG. First, in the domain sequence of the modified spider silk fibroin shown in FIG. 5 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “most on the C-terminal side”. Since it is included in the "sequence obtained by excluding the sequence from the located (A) _n motif to the C end 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 for this is 7, and s is 7 × 3 = 21. _{Similarly, all REPs are "sequences 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" (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 spider silk fibroin of FIG.

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

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 _{n motif (modified spider fibroin or natural In the derived spider fibroin), the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In all REPs contained in, 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 further. (A) The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the _{n motif.} In the calculation of the glutamine residue content, the _{reason why "the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified spider silk fibroin had its domain sequence deleted of one or more glutamine residues in the REP as compared to naturally occurring spider silk fibroin, or was replaced with another amino acid residue. It may have an amino acid sequence corresponding to the above.

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). it 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.

In the sixth modified spider silk fibroin, the hydrophobicity of REP is preferably −0.8 or more, more preferably −0.7 or more, further preferably 0 or more, and 0. It is even more preferably 3 or more, and particularly preferably 0.4 or more. 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 _{n motif (modified spider fibroin or natural In the derived spider fibroin), the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In all REPs contained in, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the _{sequence from the n} motif located most on the C-terminal side to the C-terminal of the domain sequence is taken from the domain sequence. The degree of hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the (A) _{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 closest to the 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 spider fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring spider fibroin, and / or one or more in REP. In addition to the modification corresponding to the replacement of the glutamine residue in the above with another amino acid residue, the modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues There may be.

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

As more specific examples of the sixth modified spider silk fibroin, (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917). Alternatively, modified spider silk fibroin containing the amino acid sequence set forth in SEQ ID NO: 42 (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30. , A modified spider silk fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

The modified spider silk fibroin of (6-i) will be described. The amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting VL for all 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. The amino acid sequence shown in SEQ ID NO: 27 is one in which all QQs in the amino acid sequence shown in 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 QQs in the amino acid sequence (Met-PRT525) shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 31 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.

In the amino acid sequence shown by 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.

In the amino acid sequence (Met-PRT917) shown in SEQ ID NO: 41, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with LI, and the remaining Qs are replaced with V. In the amino acid sequence (Met-PRT1028) shown in SEQ ID NO: 42, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with IF, and the remaining Qs are replaced with T.

The amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all residual glutamine. The group content is 9% or less (Table 2).

The modified spider silk fibroin of (6-i) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It may consist of the amino acid sequence shown by 42.

The modified spider silk fibroin of (6-ii) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 42. The modified spider silk fibroin of (6-ii) 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. The sequence identity is preferably 95% or more.

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

The sixth modified spider silk 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 spider silk fibroin.

As a more specific example of the modified spider silk fibroin containing a 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: Modified spider silk comprising the amino acid sequence set forth in No. 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028). Fibroin, or (6-iv), 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, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. Examples thereof include modified spider silk fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence.

The amino acid sequences shown 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, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are, respectively, SEQ ID NO: 25. , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 shown by SEQ ID NO: 11 at the N-terminal of the amino acid sequence. The amino acid sequence (including His tag sequence and hinge sequence) is added. 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. , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

The modified spider silk fibroin of (6-iii) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: It may consist of the amino acid sequence shown by 44.

The modified spider silk fibroin of (6-iv) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in No. 44. The modified spider silk fibroin of (6-iv) 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. The sequence identity is preferably 95% or more.

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

The sixth modified spider silk 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 spider silk fibroin includes a first modified spider silk fibroin, a second modified spider silk fibroin, a third modified spider silk fibroin, a fourth modified spider silk fibroin, a fifth modified spider silk fibroin, and a sixth modified spider silk fibroin. Among the characteristics of the modified spider silk fibroin, the modified spider silk fibroin may have at least two or more characteristics.

The modified spider silk fibroin may be hydrophilic modified spider silk fibroin or hydrophobic modified spider silk fibroin. In the present specification, the term "hydrophilic modified spider silk fibroin" refers to the sum of the hydrophobicity indexes (HI) of all the amino acid residues constituting the modified spider silk fibroin, and then the sum is the total number of amino acid residues. It is a modified spider silk fibroin having a value divided by (mean HI) of 0 or less. The hydrophobicity index is as shown in Table 1. Further, the modified spider silk fibroin having an average HI of more than 0 may be referred to as a hydrophobic modified spider silk fibroin.

Examples of the hydrophilic modified spider silk fibroin include the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, and sequence. The amino acid sequence represented by No. 14 or SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. Examples thereof include modified spider silk fibroin containing the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

Examples of the hydrophobic modified spider silk fibroin include the amino acid sequence and SEQ ID NO: shown by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43. 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, Modified spider silk fibroin containing the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 44.

The modified spider silk fibroin is preferably a modified spider silk fibroin having an average value (mean HI) of the hydrophobicity index of -1.0 or more, preferably -0.9 or more, from the viewpoint of being more excellent in water retention. More preferably, it is a modified spider silk fibroin. The average HI of the modified spider silk fibroin is, for example, -0.8 or more, -0.7 or more, -0.6 or more, -0.5 or more, -0.4 or more, -0.3 or more, -0. 2 or more, -0.1 or more, 0 or more, 0.1 or more, 0.2 or more, or 0.3 or more, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 Hereinafter, it may be 1.1 or less, 1.0 or less, or 0.9 or less.

The modified spider silk fibroin having an average HI of −1.0 or higher may be, for example, the above-mentioned hydrophobic modified spider silk fibroin, or a hydrophilic modified spider silk fibroin having an average HI of −1.0 or higher. You may. Examples of the modified spider silk fibroin having an average HI of -1.0 or more include the fourth fibroin, SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), and the fifth fibroin, SEQ ID NO: 22 (PRT720). , SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), the sixth fibroin, SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), Modified spider silk fibroin containing the amino acid sequence set forth in SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966) or SEQ ID NO: 43 (PRT917) or the above SEQ ID NO: Examples thereof include an amino acid sequence having 90% or more homology with the indicated amino acid sequence.

The modified spider silk fibroin according to the present embodiment can be produced by a conventional method using a nucleic acid encoding the modified spider silk fibroin. The nucleic acid encoding the modified spider silk fibroin may be chemically synthesized based on the base sequence information, or may be synthesized by using a PCR method or the like. In addition, the produced modified spider silk fibroin can be isolated and purified by a commonly used method.

(Water retention agent)
The water-retaining agent according to the present embodiment may contain one type of modified spider silk fibroin alone, or may contain two or more types of modified spider silk fibroin in combination.

The water-retaining agent according to the present embodiment may further contain other additives (ingredients other than the active ingredient) depending on its form, use and the like. Additives include, for example, plasticizers, leveling agents, cross-linking agents, crystal nucleating agents, antioxidants, UV absorbers, colorants, fillers, and synthetic resins. The content of the additive may be 50 parts by mass or less with respect to 100 parts by mass of the total amount of the water retention-imparting agent.

The water-retaining agent according to the present embodiment may be in any form of, for example, powder, paste, liquid (for example, suspension, solution), or pellet. The water retention according to the present embodiment may also be in the form of a molded product such as a fiber, a film, a gel, a porous body, or particles. The water-retaining form according to the present embodiment may be appropriately set according to the object to which water-retaining is applied (water-retaining article) and its use.

Since the water-retaining agent according to the present embodiment contains modified spider silk fibroin as a main component, it can be molded into any of the above-mentioned forms. The molded product may be a molded product obtained by molding the modified spider silk fibroin itself, or may be molded by combining the modified spider silk fibroin with another material.

When the water-retaining agent according to the present embodiment is prepared in the form of powder, for example, the protein obtained by the above-mentioned method for producing modified spider silk fibroin may be dried and made into powder. If necessary, the protein powder may contain other additives, and the particle size of the powder may be adjusted.

When the water-retaining agent according to the present embodiment is prepared in the form of a liquid (for example, a solution), for example, the protein obtained by the above-mentioned method for producing modified spider fibroin is dissolved in a soluble solvent to be liquid (modified). It may be spider silk fibroin solution). If necessary, the modified spider silk fibroin solution may contain other additives. Examples of the solvent capable of dissolving the modified spider fibroin include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, hexafluoroisopropanol (HFIP) and the like. An inorganic salt may be added to the solvent as a dissolution accelerator.

When the water-retaining agent according to the present embodiment is prepared in the form of a liquid (for example, suspension), the modified spider silk fibroin may be dispersed in a dispersible solvent to make it liquid. Examples of the solvent in which the modified spider silk fibroin can be dispersed include lower alcohols, water, and a mixed solution of alcohol and water.

When the water-retaining agent according to the present embodiment is prepared in the form of fibers, for example, a known spinning method such as wet spinning, dry spinning, dry wet spinning, or melt spinning, using the above-mentioned modified spider fibroin solution as a dope solution. May be spun into fibers (modified spider fibroin fibers). The form of the fiber may be a single yarn, a composite yarn such as a blended yarn, a mixed fiber yarn, a mixed weaving yarn, a mixed weaving yarn, a twisted yarn, a covering yarn, or a non-woven fabric. Good.

The modified spider silk fibroin fiber may be a short fiber or a long fiber. Further, the modified spider silk fibroin fiber may be the modified spider silk fibroin fiber alone or in combination with other fibers. That is, a single yarn composed of only modified spider silk fibroin fibers and a composite yarn composed of a combination of modified spider yarn fibroin fibers and other fibers may be used alone or in combination thereof. The single yarn and the composite yarn may be spun yarns in which short fibers are twisted, or filament yarns in which long fibers are twisted or not twisted. Further, the modified spider silk fibroin fiber, whether it is a short fiber or a long fiber, can be used as a fiber alone or in combination with other fibers without being processed into a yarn. Examples of other fibers include synthetic fibers such as nylon and polyester, recycled fibers such as cupra and rayon, and natural fibers such as cotton and linen. When used in combination with other fibers, the content of the modified spider silk fibroin fiber is preferably 20% by mass or more, more preferably 30% by mass or more, based on the total amount of fibers, 40. It is more preferably mass% or more, and even more preferably 50 mass% or more.

When the water-retaining agent according to the present embodiment is prepared in the form of a film, gel, porous body, particles, etc., for example, Japanese Patent Application Laid-Open No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735, etc. It can be manufactured according to the described method.

[Method of imparting water retention to articles]
The method for imparting water retention to an article according to the present embodiment includes a step of incorporating the modified spider silk fibroin into the article. Since the modified spider silk fibroin according to the present invention has excellent water retention, it is possible to impart water retention to the article by incorporating it in the article.

The article is not particularly limited as long as it should be provided with water retention. Specifically, fibers, woven fabrics, knitted fabrics, non-woven fabrics, cotton, sponges, films, resins, composite materials (regardless of the form of the water-retaining agent according to the present embodiment), and various types produced using them. Goods and the like can be mentioned.

The step of containing the modified spider silk fibroin may be a step of incorporating the modified spider silk fibroin by incorporating the water-retaining agent according to the present invention described above. The modified spider silk fibroin may be contained in an article in the form of, for example, long fibers, short fibers, gels, particles, porous bodies, films, etc., and the article may be coated with liquid modified spider silk fibroin or the article may be coated. It may be contained in an article by impregnating it with liquid modified spider silk fibroin.

The method for containing the modified spider silk fibroin is not particularly limited, and a method of mixing the modified spider silk fibroin with the material (raw material) may be used. It may be a method of forming an article in combination with the material (molded body or the like) of. Further, it may be a method of forming an article (molded article) by molding the modified spider silk fibroin (which may contain other additives if necessary) itself.

Specifically, for example, fibers may be formed by spinning using a dope solution obtained by adding a powdery water-retaining agent to a solvent in which modified spider silk fibroin can be dissolved, and by casting. A film may be formed, and a gel, particles or a porous body may be formed. Further, the raw material may be mixed with a water-retaining agent in the form of powder or pellets and melt-spun to form fibers, molded products and the like. Alternatively, a composite resin may be formed by mixing the resin with short fibers.

The content of the modified spider silk fibroin in the article is preferably 20% by mass or more, more preferably 30% by mass or more, further preferably 40% by mass or more, based on the total amount of the article, 50% by mass or more. It is even more preferably mass% or more. The upper limit of the content of the modified spider silk fibroin may be 100% by mass or 90% by mass or less based on the total amount of the article. By adjusting the content of the modified spider silk fibroin in the article, the water retention of the article can be controlled. That is, since the modified spider silk fibroin according to the present invention is excellent in water retention, the water retention of the article can be further increased as the content of the modified spider silk fibroin in the article is increased.

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

[Manufacturing of modified spider silk fibroin]
(1) Preparation of expression vector Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes, a modified spider silk fibroin having SEQ ID NO: 15 (1) Hereinafter, a modified spider silk fibroin (hereinafter, also referred to as “PRT966”) having a “PRT799”) and SEQ ID NO: 40 was designed.

The amino acid sequence shown in SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes, and further. The amino acid sequence (tag sequence and hinge sequence) shown by SEQ ID NO: 11 is added to the N-terminal.

The amino acid sequence shown in SEQ ID NO: 40 is a sequence in which the regions of 20 domain sequences existing in the amino acid sequence (Met-PRT410) shown in SEQ ID NO: 7 are repeated twice for the purpose of improving the hydrophobicity. The amino acid sequence shown by SEQ ID NO: 11 is further added to the N-terminal to which all QQs are replaced with VF and the remaining Qs are replaced with I (amino acid sequence shown by SEQ ID NO: 32).

The average hydrophobicity index (average HI) of the modified spider silk fibroin PRT799 is −0.801, and the average hydrophobicity index (average HI) of the modified spider silk fibroin PRT966 is 0.466.

Next, nucleic acids encoding the designed modified spider silk fibroin PRT799 and PRT966 were synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.

(2) Expression of modified spider silk fibroin Escherichia coli BLR (DE3) was transformed with the expression vector obtained in (1). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin 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. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured under constant pH 6.9. Further, 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 maintained at 37 ° C., and the cells were cultured 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 modified spider silk fibroin. Twenty hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture broth before and after the addition of IPTG, and the appearance of the target recombinant structural protein band dependent on the addition of IPTG resulted in the expression of the target modified spider silk fibroin. It was confirmed.

(3) Purification of modified spider silk fibroin The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The 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 at 60 ° C. Was stirred with a stirrer for 30 minutes 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 agglutinating protein obtained after dialysis was recovered by centrifugation, water was removed by a freeze-dryer, and the freeze-dried powder was recovered to obtain modified spider silk fibroin (PRT799 and PRT966).

[Manufacturing of modified spider silk fibroin fiber]
(1) Preparation of Dope Solution The powdered spider silk fibroin (PRT799 or PRT966) and formic acid (purity 99%) obtained in the above purification step were mixed and stirred at room temperature to dissolve them. A dope solution was prepared by filtering with a metal filter having a mesh size of 1 μm. The contents of formic acid and modified spider silk fibroin in each doping solution were 70% by weight (formic acid) and 30% by weight (modified spider silk fibroin), respectively, with the total amount of the doping solution being 100% by weight.

(2) Wet Spinning Using a known spinning apparatus, each of the doping liquids prepared in (1) above was discharged into the coagulating liquid by a gear pump to perform wet spinning. The spinning conditions were as shown below. As a result, a modified spider silk fibroin fiber was obtained.
(Spinning conditions)
Spinning nozzle hole diameter: 0.3 mm
Coagulant: 18% by mass sodium sulfate aqueous solution Coagulant temperature: 40 ° C
Water wash bath temperature: 90 ° C
Stretching ratio in water washing bath: 3.4 times Hot roller temperature: 60 ° C

(3) Shrinkage treatment The modified spider silk fibroin fiber obtained in (2) was shrink-proofed according to the following procedure. The fibers were cut to a length of about 60 cm and bundled in plurality to obtain a fiber bundle having a fineness of 150 denier. The fiber bundle was immersed (wet) in water at 90 ° C. for 1 hour to remove residual stress in the fiber derived from the spinning process, and allowed to stand overnight at room temperature to dry.

[Evaluation of water retention of modified spider silk fibroin]
The water retention was evaluated by the following procedures (1) to (7). The number of samples was n = 3. For comparison, polyester fiber (manufactured by Jig Co., Ltd., model number 5993) and natural silk fiber (commercially available product, fiber diameter 15 μm) were prepared.
(1) Each fiber was cut into a length of 15 cm.
(2) Calculate the value of the weight per fiber and the side area from the weight per 50 fibers, adjust the number of fibers so that ^{the sum of the side areas of the fiber bundles is 500 cm 2, and prepare each sample.} Made. The side area was calculated on the assumption that the fiber diameter was measured and the fiber cross section was circular.
(3) Each sample was allowed to stand for 24 hours or more in a constant temperature and humidity chamber set at a temperature of 20 ° C. and a relative humidity of 65%.
(4) The weight of each sample after curing (standing) was measured and used as the initial weight ( _Wi ).
(5) Each sample was impregnated in tap water at 25 ° C. for 15 minutes, then divided into small portions in a washing net and dehydrated in a washing machine for 15 minutes.
(6) After measuring the weight of each sample immediately after dehydration (0 hours), the sample is allowed to stand in a constant temperature and humidity chamber set at a temperature of 20 ° C. and a relative humidity of 65%, and the weight (W) of each sample is measured every hour. Was measured for a total of 8 hours.
(7) The water content was calculated from the weight of each sample at each time, and the water retention was evaluated.
The evaluation results are shown in Table 6, Table 7, and FIG. The water content [g] shown in Table 6 and the ratio [%] of the water content per fiber weight shown in Tables 7 and 6 were calculated from the following formulas (a) and (b).
Formula (a): Water content [g] = Sample weight W [g] at each time-Initial sample weight _Wi [g]
Formula (b): Ratio of water content per fiber weight [%] = water content [g] / initial weight of sample ( _Wi ) [g] × 100

As shown in Tables 6, 7 and 6, the fibers containing the modified spider silk fibroin (Examples 1 and 2) can retain water for a long time, have excellent water retention ability, and have an initial water retention amount. It was confirmed that it was excellent at (0 hours) and had extremely excellent water retention. On the other hand, the natural silk fiber (Comparative Example 1) is excellent in the initial water retention capacity but inferior in the water retention capacity, and the polyester fiber (Comparative Example 2) is significantly inferior in both the initial water retention capacity and the water retention capacity. It was confirmed that. For example, the water retention can be improved by incorporating the modified spider silk fibroin in a synthetic resin fiber or the like as a water retention imparting agent.

Claims (8)

A water-retaining agent containing modified spider silk fibroin as an active ingredient. The water retention-imparting agent according to claim 1, wherein the modified spider silk fibroin contains a modified spider silk fibroin having an average hydrophobicity index of −1.0 or higher. The water retention-imparting agent according to claim 1 or 2, which is in the form of powder, liquid or fiber. The water-retaining agent according to any one of claims 1 to 3, which is in the form of a fiber. A method of imparting water retention to an article
A method comprising the step of incorporating modified spider silk fibroin into the article. The method of claim 5, wherein the modified spider silk fibroin comprises a modified spider silk fibroin having an average hydrophobicity index of −1.0 or higher. The method of claim 5 or 6, which is in the form of powder, liquid or fiber. The method according to any one of claims 5 to 7, which is in the form of a fiber.

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