JP7674739B2 - Modified fibroin - Google Patents

Description

The present invention relates to modified fibroins.

Fibroin is a type of fibrous protein that contains up to 90% of glycine, alanine and serine residues that lead to the formation of β-pleated sheets (Non-Patent Document 1). Known examples of fibroin include proteins that constitute the silk produced by insects and spiders (silk proteins, hornet silk proteins, spider silk proteins, etc.).

Silk protein has excellent mechanical properties, moisture absorption properties, and deodorizing properties, and is widely used as a material for clothing. Silk thread is also used for surgical sutures, etc., because it is a natural fiber with immune tolerance and high biocompatibility.

Spiders have up to seven types of silk glands, each producing fibroin (spider silk protein) with different properties. Spider silk proteins are named according to their source organs: major ampullate spider protein (MaSp), which has high toughness, minor ampullate spider protein (MiSp), which has high elongation force, and flagelliform (Flag), tubuliform, aggregate, aciniform, and pear-shaped (pyriform) spider silk proteins. In particular, structural research has been intensively conducted on major ampullate spider protein, which has high toughness due to its excellent strength and elongation (Patent Document 1 and Patent Document 2).

One of the structures specific to fibroin is a repeat structure of amino acid motifs classified as GPGXX, an alanine residue-rich extension region ((A) _n or (GA) _n ), GGX, and a spacer (Non-Patent Document 2). It has also been reported that the replacement of the (GA) _n motif with the (A) _n motif reduces the elongation but increases the tensile strength, that the elongation increases by increasing the number of GPGXX motifs, and that the tensile strength increases by replacing some of the GPGXX motifs with the (A) _n motif (Patent Document 2). The GGX and GPGXX motifs are also thought to form a flexible helical structure that gives elasticity to the thread (Patent Document 3).

Recombinant spider silk proteins and recombinant silk proteins have been produced in several heterologous protein production systems. For example, many reports have been published on recombinant fibroin production using recombinant protein production systems with hosts such as goats, silkworms, plants, mammalian cells, yeasts, molds, gram-negative bacteria, and gram-positive bacteria, and certain results have been obtained (Non-Patent Document 3, Patent Documents 4 and 5).

In recent years, the demand for heat-resistant materials has been increasing in various industrial fields, including automobiles, electrical and electronics. Silk, which is made of fibroin, has an elegant texture, beautiful luster, and is comfortable to wear, but it has drawbacks such as shrinking and yellowing when wet, and it is recommended to iron it at a medium temperature of up to 160°C. It is generally considered to be a fiber with low heat resistance. In order to use it as a material for heat-resistant composite materials, it is first necessary to improve the heat resistance of the silk protein itself.

JP 2012-55269 A Special Publication No. 2005-502347 Special Publication No. 2009-505668 Special table 2014-502140 publication International Publication No. 2015/042164

Asakura et al., Encyclopedia of Agricultural Science, Academic Press: New York, NY, 1994, Vol. 4, pp. 1-11 Microbial Cell Factories, 2004, 3:14 Science, 2002, Vol. 295, pp. 472-476

Natural spider silk made of fibroin has excellent properties against physical changes such as tensile strength, toughness, and extensibility, has high biocompatibility and biodegradability, and also has excellent heat resistance. However, unlike silkworms, which use natural silk industrially, it is difficult to mass-produce spiders, and therefore attempts have been made to produce artificial synthetic fibers that imitate spider silk and apply them to materials and ingredients such as silk and bedding that have the above-mentioned properties against physical changes, as well as biocompatibility and biodegradability, but no artificial fibers comparable in heat resistance to natural spider silk have been successfully produced.

Therefore, an object of the present invention is to provide a modified fibroin having improved thermal stability.

The present inventors have found that by increasing the alanine content of the alanine consecutive sequence in the (A) _n motif present in fibroin, a fibroin having improved thermal stability can be obtained. The present invention is based on this novel finding.

That is, the present invention relates to, for example, the following inventions.

[1]

A modified fibroin comprising a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m -(A) _n motif,

(A) the total number of 6 or more consecutive alanine residues in _{the n} motif is 20% or more of the total number of all amino acid residues;

The hydrophobicity of the REP is −1.0 or more;

A modified fibroin having a total number of amino acid residues of 580 or more.

[In formula 1 and formula 2, (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.]

[2]

The modified fibroin according to [1], wherein the hydrophobicity of the REP is 0 or more.

[3]

The modified fibroin according to [1] or [2] above, wherein the total number of consecutive alanine residues in _{the n} motif (A) is 20% or more of the total number of amino acid residues.

[4]

A modified fibroin comprising an amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[5]

The modified fibroin according to any one of [1] to [4], further comprising a tag sequence at either or both of the N-terminus and the C-terminus.

[6]

The modified fibroin according to [5], wherein the tag sequence comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5.

[7] A modified fibroin comprising an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[8]

The modified fibroin according to any one of [1] to [7], having a thermal decomposition temperature (Td) of 268°C or higher.

[9]

A nucleic acid encoding the modified fibroin according to any one of [1] to [8].

[10]

A nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid according to [9] and codes for a modified fibroin comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. [In formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.]

[11]

A nucleic acid encoding a modified fibroin having a sequence identity of 90% or more with the nucleic acid according to [9] and comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif.

[In formula 1 and formula 2, (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.]

[12]

A method for producing modified fibroin, comprising the steps of:

expressing the nucleic acid by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence;

A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin described in any one of [1] to [8].

[13]

A method for improving the thermal stability of a modified fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m -(A) _n motif, comprising:

The method includes increasing the number of consecutive alanine residues in at least one (A) _n motif by inserting one or more alanine residues into at least one (A) _n motif or REP in the unmodified fibroin or by substituting another amino acid residue adjacent to the alanine residue with alanine;

The method comprises the step of: the thermal decomposition temperature (Td) of the modified fibroin is 5°C or more higher than that of the unmodified fibroin.

[In formula 1 and formula 2, (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.]

[14]

The method according to [13], wherein the unmodified fibroin is naturally occurring fibroin.

[15]

The modified fibroin according to [14], wherein the naturally occurring fibroin is fibroin derived from an insect or arachnid.

[16]

The modified fibroin according to [15], wherein the naturally occurring fibroin is a large ampullate spider protein (MaSp) or a small ampullate spider protein (MiSp) of an arachnid.

[17]

The modified fibroin according to any one of [1] to [8] is included,

1. An article of manufacture selected from the group consisting of fibers, yarns, films, foams, granules, nanofibrils, gels and resins.

According to the present invention, it is possible to provide a modified fibroin having improved thermal stability.

FIG. 1 is a schematic diagram showing an example of a domain arrangement of spider fibroin. 1 is a graph showing the results of comparing the thermal decomposition temperatures of PRT380 and PRT525. 1 is a graph showing the results of comparing the glass transition points of PRT380 and PRT525. 1 is a graph showing the results of comparing the thermal decomposition temperatures of PRT1068, PRT1069, and PRT1070. 1 is a graph showing the results of comparing the thermal decomposition temperatures of PRT525 and PRT1070.

The following describes in detail the form for implementing the present invention. However, the present invention is not limited to the following embodiment.

[Modified fibroin] The modified fibroin according to the present invention is a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. The modified fibroin may further have amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but are not limited to, regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of about 100 amino acid residues.

As used herein, "modified fibroin" refers to a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin. As used herein, "naturally occurring fibroin" refers to a fibroin whose amino acid sequence is identical to that of fibroin produced by naturally occurring insects, arachnids, etc. Naturally occurring fibroin is also a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif.

A "modified fibroin" may be one whose amino acid sequence has been modified based on naturally occurring fibroin (e.g., one whose amino acid sequence has been modified by modifying the genetic sequence of a cloned naturally occurring fibroin) so long as it has the amino acid sequence specified in the present invention, or one whose amino acid sequence has been artificially designed and synthesized without relying on naturally occurring fibroin (e.g., one having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence). Note that modified fibroin whose amino acid sequence has been modified is also included in the modified fibroin, so long as the amino acid sequence differs from that of naturally occurring fibroin.

In the present specification, the term "domain sequence" refers to an amino acid sequence that generates a crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically corresponding to REP of the amino acid sequence) specific to fibroin, and means an amino acid sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif. Here, the (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 4 to 27. The number of amino acid residues in the (A) _n motif may be 4 to 20, 8 to 20, 10 to 20, 4 to 16, 5 to 10, 6 to 12, 6 to 18, 7 to 10, 7 to 14, 8 to 16, or 11 to 16. In addition, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may have the same amino acid sequence as each other or different amino acid sequences.

The (A) _n motif may have a number of alanine residues of 80% or more, preferably 85% or more, more preferably ₉₀ % or more, even more preferably 95% or more, and even more preferably 100% (meaning composed only of alanine residues). Of the (A) _n motifs present in a plurality in the domain sequence, at least seven are preferably composed only of alanine residues. "Composed only of alanine residues" means that the (A) _n motif has an amino acid sequence represented by (Ala) _k (Ala represents an alanine residue, and k represents an integer of 4 to 27, preferably an integer of 4 to 20, more preferably an integer of 4 to 16).

The modified fibroin of this embodiment has (A) an _n motif in which the total number of consecutive alanine residues of 6 or more is 20% or more of the total number of amino acid residues, the hydrophobicity of REP is -1.0 or more, and the total number of amino acid residues is 580 or more.

In the modified fibroin according to the present embodiment, the total number of 6 or more consecutive alanine residues in the (A) _n motif is 20% or more of the total number of amino acid residues. The total number of 6 or more consecutive alanine residues in the (A) _n motif means the total number of alanine residues when only the number of 6 or more consecutive alanine residues is counted. Therefore, the number of 5 consecutive alanine residues is not counted. In addition, in the (A) _n motif, when there are no 6 or more consecutive alanine residues due to the presence of other amino acid residues between the alanine residues, the number of the alanine residues is not counted.

The total number of 6 or more consecutive alanine residues in the (A) _n motif may be, for example, 21% or more, 22% or more, or 23% or more of the total number of all amino acid residues. Also, the total number of 7 or more consecutive alanine residues in the (A) _n motif may be, for example, 20% or more, 21% or more, 22% or more, or 23% or more of the total number of all amino acid residues.

The modified fibroin of this embodiment has a total number of amino acid residues of 580 or more. The total number of amino acid residues of the entirety may be, for example, 590 or more, 600 or more, 610 or more, or 620 or more.

The modified fibroin according to this embodiment preferably contains at least one motif selected from the GGX motif and the GPGXX motif (wherein G represents a glycine residue, P represents a phenylalanine residue, and X represents an amino acid residue other than a glycine residue) in the amino acid sequence of REP. By containing these motifs in REP, the extensibility of the modified fibroin can be improved.

When the modified fibroin of this embodiment contains a GPGXX motif in the REP, the GPGXX motif content is typically 1% or more, may be 5% or more, and is preferably 10% or more. This allows the elongation of the modified fibroin to be further improved. There is no particular upper limit to the GPGXX motif content, and it may be 50% or less, or 30% or less.

In this specification, "GPGXX motif content" is a value calculated by the following method.

In a fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif, the GPGXX motif content is calculated as x/y, where x is three times the total number of GPGXX motifs contained in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence (i.e., equivalent to the total number of G and P in the GPGXX motif), and y is the total number of amino acid residues in all REPs contained in the domain sequence obtained by excluding from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence _and further excluding the (A)n motif.

In calculating the GPGXX motif content, the target is "the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because the "sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" (sequence corresponding to REP) may contain a sequence that has low correlation with the sequence characteristic of fibroin, and when m is small (i.e., when the domain sequence is short), this affects the calculation result of the GPGXX motif content, and this influence is to be eliminated. Note that when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 1 is a schematic diagram showing the domain sequence of a modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 1. First, in the domain sequence of the modified fibroin shown in FIG. 1 (which is of the "[(A) _n motif-REP] _m -(A) _n motif" type), all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 1), so the number of GPGXX motifs for calculating x is 7, and x is 7×3=21. Similarly, all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 1), so the total number y of amino acid residues of all REPs obtained by further removing the (A) _n motif from the sequence is 50+40+10+20+30=150. Next, x/y (%) can be calculated by dividing x by y, which is 21/150=14.0% in the case of the modified fibroin of FIG.

The modified fibroin of this embodiment preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%. This allows the effects of the present invention to be more pronounced.

In this specification, "glutamine residue content" is a value calculated by the following method.

In a fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif, in all REPs contained in a sequence (sequence corresponding to "region A" in Figure 1) obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence, the total number of glutamine residues contained in the region is w, and the total number of amino acid residues of all REPs removed from the domain sequence from the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence and further excluding the (A) _n motif is y, the glutamine residue content is calculated as w/y. The reason for targeting "the sequence removed from the domain sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" in calculating the glutamine residue content is the same as that described above.

The modified fibroin of this embodiment may have an amino acid sequence in which the domain sequence is such that, compared to the fibroin before modification, one or more glutamine residues in REP have been deleted or replaced with other amino acid residues.

The "other amino acid residue" may be any amino acid residue other than glutamine residue, but is preferably an amino acid residue with a higher hydrophobicity index than glutamine residue. The hydrophobicity index of amino acid residues is determined by a known index (Hydrophathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as "HI") of each amino acid is as shown in Table 1 below.

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

In the modified fibroin of this embodiment, the hydrophobicity of the REP may be -1.0 or more, preferably -0.9 or more, more preferably -0.8 or more, even more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.2 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of the REP, and it may be, for example, 1.0 or less, or 0.7 or less. The hydrophobicity of the REP may be, for example, -1.0 or more and 1.0 or less, or 0 or more and 0.7 or less.

In this specification, "hydrophobicity of REP" is a value calculated by the following method.

In a fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A)n motif, in all REPs contained in the sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence (the sequence corresponding to "region A" in Figure 1), the sum of the hydrophobicity indexes of each amino acid residue in the region is z, and the total number of amino acid residues in all REPs removed from the domain sequence from the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence and further excluding the (A) _n motif is y, the hydrophobicity of the REP is calculated as z/y. The reason for targeting "the sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" in calculating the hydrophobicity of the REP is the same as that described above.

The modified fibroin of this embodiment is obtained by calculating the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin and then dividing the sum by the total number of amino acid residues (average HI), which is preferably -1.0 or more, more preferably -0.9 or more, even more preferably -0.8 or more, even more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.2 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the average HI of the modified fibroin, and it may be, for example, 1.0 or less, or 0.7 or less. The average HI of the modified fibroin may be, for example, -1.0 or more and 1.0 or less, or 0 or more and 0.7 or less.

Naturally derived fibroin is a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m -(A) _n motif, and specific examples thereof include fibroins produced by insects or arachnids.

Fibroin produced by insects includes, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia cynthia ricini, Samia cynthia, Caligra japonica, Antheraea mylitta, and Antheraea japonica. and hornet silk protein excreted by larvae of hornet (Vespa simillima xanthoptera).

A more specific example of fibroin produced by insects is silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).

Fibroin produced by spiders includes, for example, spiders belonging to the Araneus genus, such as the Japanese raven spider, the Japanese garden raven spider, the red raven spider, the green raven spider, and the Japanese bean raven spider; spiders belonging to the Neoscona genus, such as the Japanese mountain raven spider, the Japanese house raven spider, and the Japanese Satsuma spider; spiders belonging to the Pronus genus, such as the Japanese dwarf raven spider; spiders belonging to the Cyrtarachne genus, such as the Japanese spine spider and the Japanese giant spine spider; spiders belonging to the Gaster genus, such as the Japanese spine spider and the Japanese spine spider; spiders belonging to the genus Acantha, spiders belonging to the genus Ordgarius such as Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Argiope such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Arachnura such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Acusilas such as Orbweaver Spider, spiders belonging to the genus Cytophora such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Poltys such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the bush spider, the four-headed bush spider, the round bush spider, and the black bush spider, and spiders belonging to the genus Chorizopes, such as the Japanese bush spider, as well as spiders belonging to the genus Tetragnatha, such as the long-legged spider, the long-legged spider, the broad-legged spider, and the scale-like long-legged spider, spiders belonging to the genus Leucauge, such as the large white-legged spider, the medium-legged spider, and the small white-legged spider, spiders belonging to the genus Orb Weaver, such as the golden orb spider and the giant orb spider, Examples of spider silk proteins include those produced by spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as the golden spider, spiders belonging to the genus Dyschiriognatha such as the small long-legged spider, spiders belonging to the genus Latrodectus such as the black widow spider, the redback spider, the gray widow spider and the three-spotted latrodectus, and spiders belonging to the family Tetragnathiidae such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

Specific examples of fibroins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-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 accession numbers AAC04504 (amino acid sequence), U37520 (base sequence)), major angu11ate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [derived from Eurosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [derived from Eurosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephila cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

More specific examples of naturally occurring fibroin include fibroins whose sequence information is registered in NCBI GenBank. For example, from among the sequences registered in NCBI GenBank that contain INV as a division, it can be confirmed by extracting sequences in which spidroin, amplify, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION, a specific product character string from CDS, and a specific character string in TISSUE TYPE from SOURCE.

When fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method illustrated above, 663 types of fibroins (of which, 415 types were spider-derived fibroins) were extracted. Of all the extracted fibroins, there were 129 types of naturally derived fibroins containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m -(A) _n motif. Of these, the six types of naturally derived fibroins shown in Table 2 below had a GPGXX motif content of 10% or more as calculated by the above-mentioned method. The glutamine residue content of all of the six naturally derived fibroins shown in Table 2 was 9.2% or more.

The modified fibroin according to the present embodiment can be obtained, for example, by adding a modification equivalent to inserting one or more alanine residues into the (A) _n motif or REP in the domain sequence of the fibroin before modification, or substituting another amino acid residue adjacent to the alanine residue with alanine. Furthermore, the amino acid sequence may be modified equivalent to substituting, deleting, inserting and/or adding one or more amino acid residues. Substitution, deletion, insertion and/or addition of amino acid residues can be performed by a method well known to those skilled in the art, such as partial directed mutagenesis. Specifically, it can be performed according to the method described in literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

The modified fibroin according to the present embodiment can be obtained, for example, by adding a mutation to the gene sequence of a cloned naturally-occurring fibroin, which corresponds to inserting one or more alanine residues into the (A) _n motif or REP, or substituting other amino acid residues adjacent to the alanine residue with alanine. It can also be obtained, for example, by designing an amino acid sequence corresponding to inserting one or more alanine residues into the (A) _n motif or REP, or substituting other amino acid residues adjacent to the alanine residue with alanine, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

The molecular weight of the modified fibroin of the present invention is not particularly limited, and may be, for example, 40 kDa or more and 700 kDa or less. The molecular weight of the modified fibroin of the present invention may be, for example, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, and may be 600 kDa or less, 500 kDa or less, 400 kDa or less, 300 kDa or less, or 200 kDa or less.

More specific examples of the modified fibroin of the present invention include (i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or (ii) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

The modified fibroin (i) will be described. The amino acid sequence (Met-PRT1069) shown in SEQ ID NO: 1 is based on the base sequence and amino acid sequence of the naturally-occurring fibroin Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), and is modified by increasing the number of consecutive alanine residues in the amino acid sequence of consecutive alanine residues in the (A) _n motif to six, substituting all QQ with VF, and substituting the remaining Q with I, and the hydrophobicity of REP is 0.21.

The amino acid sequence shown in SEQ ID NO:2 (Met-PRT1070) is obtained by increasing the number of consecutive alanine residues by inserting one alanine residue in each (A) _n motif of the amino acid sequence shown in SEQ ID NO:1, and the hydrophobicity of REP is 0.21.

The amino acid sequence shown in SEQ ID NO: 3 (Met-PRT1076) is the REP of the amino acid sequence shown in SEQ ID NO: 2 in which several amino acid residues have been deleted, and the hydrophobicity of REP is 0.21.

The amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 all have a content of six or more consecutive alanines of 20% or more (Table 3).

The modified fibroin (ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The modified fibroin (ii) is also a protein comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. The sequence identity may be 80% or more, or may be 85% or more, and is preferably 95% or more.

The modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows the modified fibroin to be isolated, immobilized, detected, visualized, etc.

Examples of tag sequences include affinity tags that utilize specific affinity (binding ability, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). A His tag is a short peptide consisting of about 4 to 10 histidine residues lined up, and has the property of specifically binding to metal ions such as nickel, and can be used to isolate modified fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 (amino acid sequence including a His tag).

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

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

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

More specific examples of modified fibroins containing a tag sequence include (iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or (iv) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

The amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 has an amino acid sequence in which the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:5 (including a His tag) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, respectively.

The modified fibroin (iii) may consist of an amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

The modified fibroin (iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The modified fibroin (iv) is also a protein comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. The sequence identity may be 80% or more, or may be 85% or more, and is preferably 95% or more.

The amino acid sequences shown in SEQ ID NO:6 (PRT1069), SEQ ID NO:7 (PRT1070), and SEQ ID NO:8 (PRT1076) all have a content of 6 or more consecutive alanines of 20% or more (Table 4). In addition, the hydrophobicity of REP in each of these sequences is 0.21.

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

[Thermal stability] Examples of indicators for evaluating thermal stability (heat resistance) include thermal decomposition temperature (Td) and glass transition point (Tg). The thermal decomposition temperature is the temperature at which thermal decomposition of a material begins, and the glass transition point is the temperature at which a material changes from crystal to liquid. It can be said that the higher the thermal decomposition temperature and/or glass transition point, the higher the thermal stability. The thermal decomposition temperature of a protein such as the modified fibroin of the present invention can be measured, for example, by thermogravimetric analysis, which observes the mass change of a sample accompanying heating. In addition, the glass transition point of a protein such as the modified fibroin of the present invention can be measured, for example, by differential scanning calorimetry, which is an analytical method that detects the glass transition, crystallization, and melting phenomena of a polymer as changes in heat flow. The thermal decomposition temperature and/or glass transition point can be measured, for example, using a differential thermal-thermogravimetric simultaneous measurement device (DTG-60H, manufactured by Shimadzu Corporation). For example, general properties are maintained even at high temperatures, and general properties are maintained even when exposed to high temperatures for a long period of time. The thermal stability of protein fibers such as the modified fibroin of the present invention can be measured, for example, by thermomechanical analysis (TMA), which observes the changes in physical properties of a sample that accompany long-term high-temperature treatment and measures the magnitude of resistance when a test piece is subjected to a certain elongation.

The modified fibroin in this embodiment may have a thermal decomposition temperature of 268°C or more, 270°C or more, 272°C, 280°C or more, or 290°C or more. In addition, the thermal decomposition temperature of the modified fibroin in this embodiment may be 5°C or more, 8°C or more, 10°C or more, or 12°C or more higher than that of the fibroin before modification.

The modified fibroin in this embodiment may have a glass transition point (Tg) of 185°C or more, 190°C or more, 192°C or more, or 195°C or more. The glass transition point of the modified fibroin in this embodiment may be 5°C or more, 8°C or more, 10°C or more, or 12°C or more higher than that of the fibroin before modification.

[Nucleic acid] The nucleic acid according to the present invention encodes the modified fibroin according to the present invention. Specific examples of nucleic acids include nucleic acids encoding modified fibroins containing the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or modified fibroins having the amino acid sequence (tag sequence) shown in SEQ ID NO: 4 or SEQ ID NO: 5 bound to either or both of the N-terminus and C-terminus of these amino acid sequences.

A nucleic acid according to one embodiment is a nucleic acid which hybridizes under stringent conditions with a complementary strand of a nucleic acid encoding a modified fibroin according to the present invention, and encodes a modified fibroin comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. In the modified fibroin encoded by the nucleic acid, the total number of 6 or more consecutive alanine residues in the (A) _n motif is preferably 20% or more of the total number of all amino acid residues. In addition, in the modified fibroin encoded by the nucleic acid, the hydrophobicity of REP is preferably -1.0 or more. In addition,
The modified fibroin encoded by the nucleic acid preferably has a total number of amino acid residues of 580 or more.

"Stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. "Stringent conditions" may be low stringent conditions, medium stringent conditions, or high stringent conditions. Low stringent conditions mean that hybridization occurs only when there is at least 85% or more identity between the sequences, for example, 5xSSC containing 0.5% SDS is used and hybridization is performed at 42°C. Medium stringent conditions mean that hybridization occurs only when there is at least 90% or more identity between the sequences, for example, 5xSSC containing 0.5% SDS is used and hybridization is performed at 50°C. High stringent conditions mean that hybridization occurs only when there is at least 95% or more identity between the sequences, for example, 5xSSC containing 0.5% SDS is used and hybridization is performed at 60°C.

A nucleic acid according to another embodiment is a nucleic acid encoding a modified fibroin having 90% or more sequence identity with a nucleic acid encoding a modified fibroin according to the present invention, and including a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. In the modified fibroin encoded by the nucleic acid, the total number of 6 or more consecutive alanine residues in the (A) _n motif is preferably 20% or more of the total number of all amino acid residues. In addition, in the modified fibroin encoded by the nucleic acid, the hydrophobicity of REP is preferably -1.0 or more. In addition, in the modified fibroin encoded by the nucleic acid, the total number of all amino acid residues is preferably 580 or more. The sequence identity may be 80% or more, or may be 85% or more, and is preferably 95% or more.

[Host and Expression Vector] In this embodiment, the expression vector has the nucleic acid sequence of the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence. The regulatory sequence is a sequence that controls the expression of a recombinant protein in a host (e.g., a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, etc.

The host according to the present invention is transformed with the expression vector according to the present invention. As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

As an expression vector, one that is capable of autonomous replication in a host cell or that can be integrated into a host chromosome and contains a promoter in a position where it can transcribe the nucleic acid of the present invention is preferably used.

When a prokaryote such as a bacterium is used as a host, the expression vector according to the present invention is preferably a vector capable of autonomous replication in the prokaryote and containing a promoter, a ribosome binding sequence, the nucleic acid according to the present invention, and a transcription termination sequence. A gene that controls the promoter may also be included.

Prokaryotes include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas.

Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli BL21 (Novagen), Escherichia coli BL21(DE3) (Life Technologies), Escherichia coli BLR(DE3) (Merck Millipore), Escherichia coli DH1, Escherichia coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC 23506), Escherichia coli KY3276, Escherichia coli MC1000, Escherichia coli MG1655 (ATCC 47076), and Escherichia coli No. 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 (ATCC 27325), Escherichia coli XL1-Blue, and Escherichia coli XL2-Blue.

Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus borstelensis, Brevibacillus centroporas, Brevibacillus formosus, Brevibacillus invocatus, Brevibacillus lathyrosporus, Brevibacillus limnophilus, Brevibacillus parabrevis, Brevibacillus reuszeri, Brevibacillus thermolvar, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevibacillus brevis 47-5Q (JCM8975), Brevibacillus choshinensis HPD31 (FERM Examples of the Brevibacillus choshinensis strain include Brevibacillus choshinensis HPD31-S (FERM BP-6623), Brevibacillus choshinensis HPD31-OK (FERM BP-4573), and Brevibacillus choshinensis SP3 strain (manufactured by Takara Biosciences).

Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens ATCC14460, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesi, Serratia proteamaculans, Serratia odorifera, Serratia plymuthica, and Serratia rubidaea.

Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens.

Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphylum ATCC 15354.

Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, Brevibacterium immariophilum (Brevibacterium immariophilum) ATCC14068, Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC13869) ATCC13665, ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium saccharolyticum (Brevibacterium Examples of the bacterium include Brevibacterium saccharolyticum ATCC 14066, Brevibacterium thiogenitalis ATCC 19240, Brevibacterium album ATCC 15111, and Brevibacterium serinum ATCC 15112.

Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6871, ATCC6872, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium acetoacidophilum ATCC14067, Corynebacterium acetos ... Corynebacterium acetoglutamicum ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolyticum ATCC21511, Corynebacterium callunae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, Corynebacterium lilium ATCC15990, Corynebacterium melassecora ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), Corynebacterium herculis ATCC13868, and the like can be mentioned.

Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas fulva, and Pseudomonas sp. D-0110.

Any method for introducing an expression vector into the host cell can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], a protoplast method (JP Patent Publication 63-248394), or a method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979) can be mentioned.

Transformation of microorganisms belonging to the genus Brevibacillus can be carried out, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156:1130-1134), the method of Takagi et al. (Agric. Biol. Chem., 1989, 53:3099-3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61:202-203).

Examples of vectors into which the nucleic acid according to the present invention is introduced (hereinafter simply referred to as "vectors") include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (JP Patent Publication 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., 1989, 53, 277 (1989)], pGEL2 [Proc. Natl. Acad. Sci., 1989, 53, 277 (1989)], pGEL1 ... USA, 82, 4306 (1985)], pBluescript II SK(-) (Stratagene), pTrs30 [Escherichia coli JM109/pTrS3
pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), JP-A-60-221091], pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), and pET system (Novagen).

When Escherichia coli is used as the host, suitable vectors include pUC18, pBluescriptII, pSupex, pET22b, and pCold.

Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP Patent Publication No. 2-31682), pNY700 (JP Patent Publication No. 4-278091), pHY4831 (J. Bacteriol., 1987, 1239-1245), pNU200 (Udaka Shigezo, Journal of the Japanese Society of Agricultural Chemistry, 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU2 11R2L5 (JP-A-7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58:525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177:745-749), pHT210 (JP-A-6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42:358-363), or pNCO2 (JP-A-2002-238569), which is a shuttle vector between Escherichia coli and microorganisms belonging to the genus Brevibacillus.

The promoter is not limited as long as it functions in the host cell. Examples of promoters include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. Artificially designed and modified promoters such as a promoter with two Ptrp promoters in series (Ptrp x 2), a tac promoter, a lacT7 promoter, and a let I promoter can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon has been adjusted to an appropriate distance (e.g., 6 to 18 bases). In the expression vector of the present invention, a transcription termination sequence is not necessarily required for the expression of the nucleic acid of the present invention, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

Eukaryotic hosts include, for example, yeast, filamentous fungi (molds, etc.), and insect cells.

Examples of yeasts include those belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, and Hansenula. More specifically, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Trichosporon pullulans, Schwanniomyces alluvius, Schwanniomyces occidentalis, Candida utilis, utilis, Pichia pastoris, Pichia angusta, Pichia methanolica, Pichia polymorpha, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, and the like.

When yeast is used as a host cell, the expression vector typically preferably contains an origin of replication (if amplification in the host is required) and a selectable marker for propagation of the vector in E. coli, a promoter and terminator for expression of the recombinant protein in yeast, and a selectable marker for yeast.

When the expression vector is a non-integrative vector, it is preferable that it further contains an autonomously replicating sequence (ARS), which can improve the stability of the expression vector in the cell (Myers, A.M., et al. (1986) Gene 45:299-310).

When yeast is used as a host, examples of vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, pPICZ B, etc.

The promoter is not limited as long as it can be expressed in yeast. Examples include promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP 1 promoter, pGAP promoter, pGCW14 promoter, AOX1 promoter, MOX promoter, etc.

Any method for introducing an expression vector into yeast can be used as long as it is a method for introducing DNA into yeast, such as the electroporation method (Methods Enzymol., 194, 182 (1990)), the spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), the lithium acetate method (J. Bacteriol., 153, 163 (1983)), and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

Filamentous fungi include, for example, the genus Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, Penicillium, Examples of fungi that can be used include fungi belonging to the genera Myceliophtora, Botrytis, Magnaporthe, Mucor, Metarhizium, Monascus, Rhizopus, and Rhizomucor.

Specific examples of filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus oryzae, Aspergillus sake, Aspergillus sojae, Aspergillus tubigensis, Aspergillus tumefaciens ... Aspergillus tubigensis, Aspergillus niger, Aspergillus nidulans, Aspergillus parasiticus, Aspergillus ficum, Aspergillus phoeicus, Aspergillus foetidus, Aspergillus flavus, Aspergillus fumigatus fumigatus, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma reesei, Chrysosporium lucknowense, Thermoascus, Sporotrichum, Sporotrichum cellulophilum cellulophilum, Talaromyces, Thielavia terrestris, Thielavia, Neurospora crassa, Fusarium oxysporus, Fusarium graminerum, Fusarium venenatum, Humicola insolens, Penicillium chrysogenum, Penicillium camemberti camemberti, Penicillium canescens, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium purpurogenum, Penicillium roqueforti, Myceliophtaora thermophilum, Mucor ambigus Mucor circinelloides, Mucor fragilis, Mucor hiemalis, Mucor inaequisporus, Mucor oblongiellipticus, Mucor racemosus, Mucor recurvus, Mucor saturninus, Mucor subtilismius subtilissmus, Ogataea polymorpha, Phanerochaete chrysosporium, Rhizomucor miehei, Rhizomucor pusillus, Rhizopus arrhizus, and the like.

When the host is a filamentous fungus, the promoter may be any of genes related to the glycolysis pathway, genes related to constitutive expression, and enzyme genes related to hydrolysis, and specific examples include amyB, glaA, agdA, glaB, TEF1, xynF1 tannasegene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, and catB.

Introduction of an expression vector into a filamentous fungus can be carried out using a conventional method, such as the method of Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)], the protoplast method [Mol. Gen. Genet., 168:111 (1979)], the competent method [J. Mol. Biol., 56:209 (1971)], and the electroporation method.

Examples of insect cells include lepidopteran insect cells, more specifically insect cells derived from Spodoptera frugiperda such as Sf9 and Sf21, and insect cells derived from Trichoplusia ni such as High 5.

When insect cells are used as hosts, examples of vectors include baculoviruses such as Autographa californica nuclear polyhedrosis virus, a virus that infects insects of the family Noctuidae (Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)).

When insect cells are used as hosts, the polypeptide can be expressed by the method described in, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992), Bio/Technology, 6, 47 (1988), etc. That is, a recombinant gene transfer vector and a baculovirus are co-transferred into insect cells to obtain a recombinant virus (expression vector) in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the polypeptide. Examples of gene transfer vectors used in this method include pVL1392, pVL1393, and pBlueBacIII (both manufactured by Invitrogen).

Methods for co-introducing a recombinant gene transfer vector and a baculovirus into insect cells to prepare a recombinant virus include, for example, the calcium phosphate method (JP Patent Publication No. 2-227075) and the lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).

The recombinant vector of the present invention preferably further contains a selection marker gene for selecting a transformant. For example, in E. coli, resistance genes to various drugs such as tetracycline, ampicillin, and kanamycin can be used as the selection marker gene. Recessive selection markers that can complement gene mutations involved in auxotrophy can also be used. In yeast, a resistance gene to geneticin can be used as the selection marker gene, and selection markers such as genes that complement gene mutations involved in auxotrophy, LEU2, URA3, TRP1, and HIS3 can also be used. In filamentous fungi, the selection marker genes include niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6, 386-389, (1984)), sC (Gene, 84, 329-334, (1989)), ptrA (BiosciBiotechnol Biochem, 64, 1416-1421, (2000)), pyrG (BiochemBiophys Res. Commun., 112, 284-289, (1983)), amdS (Gene, 26, 205-221, (1983)), aureobasidin resistance gene (Mol Gen Genet, 261, 290-296, (1999)), benomyl resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873, (1986)) and hygromycin resistance gene (Gene, 57, 21-26, (1987)), leucine auxotrophy complementing gene, etc. When the host is an auxotrophic mutant, a wild-type gene that complements the auxotrophy can also be used as the selection marker gene.

The selection of a host transformed with the expression vector of the present invention can be carried out by plaque hybridization, colony hybridization, etc., using a probe that selectively binds to the nucleic acid of the present invention. The probe can be a partial DNA fragment amplified by PCR based on the sequence information of the nucleic acid of the present invention and modified with a radioisotope or digoxigenin.

[Method for producing modified fibroin] The modified fibroin of the present invention can be produced by a method including a step of expressing the nucleic acid of the present invention by a host transformed with an expression vector of this embodiment. A method for producing modified fibroin of one embodiment includes a step of expressing the nucleic acid by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin of the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence.

In addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the methods described in Molecular Cloning, 2nd Edition. When expressed in yeast, animal cells, or insect cells, the modified fibroin can be obtained as a polypeptide to which sugar or a sugar chain has been added.

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

When the host of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for the host of the present invention may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host and allows the host to be cultured efficiently.

A carbon source may be any that can be assimilated by the host, and examples of carbon sources that can be used include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

Examples of nitrogen sources that can be used include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria and their digestions.

Examples of inorganic salts that can be used include potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

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

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

As culture media for insect cells, commonly used TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by Life Technologies), ExCell 400, ExCell 405 (both manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)), etc. can be used.

Insect cells can be cultured for 1 to 5 days under conditions such as a culture medium pH of 6 to 7 and a culture temperature of 25 to 30°C. Furthermore, antibiotics such as gentamicin may be added to the culture medium during culture as necessary.

When the host is a plant cell, the transformed plant cell may be cultured as it is, or may be differentiated into a plant organ and then cultured. As a medium for culturing the plant cell, a commonly used Murashige and Skoog (MS) medium, White medium, or a medium to which a plant hormone such as auxin or cytokinin has been added can be used.

Animal cells can be cultured for 3 to 60 days under conditions such as a culture medium pH of 5 to 9 and a culture temperature of 20 to 40°C. In addition, antibiotics such as kanamycin and hygromycin may be added to the medium during culture as necessary.

Methods for producing modified fibroin using a host transformed with the expression vector of this embodiment include a method for producing the modified fibroin within a host cell, a method for secreting the modified fibroin outside the host cell, and a method for producing the modified fibroin on the outer membrane of the host cell. Each of these methods can be selected by changing the host cell used and the structure of the modified fibroin to be produced.

For example, when the modified fibroin is produced inside a host cell or on the outer membrane of the host cell, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Rowe et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989); Genes Develop., 4, 1288 (1990)), or the methods described in JP-A-5-336963 and WO 94/23021 can be applied mutatis mutandis to produce the modified fibroin.
The modified fibroin can be modified so as to be actively secreted outside the host cell. That is, by using a genetic recombination technique to express a polypeptide containing an active site of the modified fibroin in a form in which a signal peptide has been added, the modified fibroin can be actively secreted outside the host cell.

The modified fibroin produced by the host transformed with the expression vector of this embodiment can be isolated and purified by a method commonly used for isolating and purifying proteins. For example, when the modified fibroin is expressed in a dissolved state within the cells, after the culture is completed, the host cells are collected by centrifugation and suspended in an aqueous buffer solution, and then disrupted by an ultrasonic homogenizer, French press, Manton Gaulin homogenizer, Dyno Mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified specimen can be obtained by using methods commonly used for isolating and purifying proteins, namely, solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, or the like, alone or in combination.

For the above chromatography, column chromatography using Phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

In addition, when the modified fibroin is expressed by forming an insoluble body within the cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the modified fibroin as a precipitate fraction. The recovered insoluble body of the modified fibroin can be solubilized with a protein denaturant. After this procedure, a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as above.

When the modified fibroin or a derivative in which a glycan has been added to the modified fibroin is secreted outside the cells, the modified fibroin or its derivative can be recovered from the culture supernatant. That is, the culture is treated by a method such as centrifugation to obtain a culture supernatant, and a purified specimen can be obtained from the culture supernatant by the same isolation and purification method as described above.

[Artificially modified fibroin composition] The artificially modified fibroin composition of this embodiment contains at least the modified fibroin of the present invention.

The content of modified fibroin in the artificial modified fibroin composition may be 30 to 100 mass%, preferably 35 to 100 mass%, and more preferably 40 to 100 mass%, based on the total amount of the artificial modified fibroin composition.

The artificial modified fibroin composition according to this embodiment may further contain other additives depending on the form, application, etc. Examples of additives include plasticizers, leveling agents, crosslinking agents, crystal nucleating agents, antioxidants, UV absorbers, colorants, fillers, and synthetic resins. The content of the additives may be 50 parts by mass or less per 100 parts by mass of the total amount of the modified fibroin.

The artificial modified fibroin composition of this embodiment may be in any form, such as a powder, a paste, or a liquid (e.g., a suspension, a solution). In addition to the form of a raw material composition (e.g., a protein powder, a dope liquid), the artificial modified fibroin composition of this embodiment may be in the form of a molded body (e.g., a fiber, a thread, a film, a foam, a granule, a molded body) that contains the artificial modified fibroin composition or that consists of the artificial modified fibroin composition.

(Dope solution) The artificial modified fibroin composition of this embodiment may be in the form of a dope solution. The dope solution of this embodiment contains at least modified fibroin and a solvent. The dope solution of this embodiment may further contain a dissolution promoter. The dope solution of this embodiment may also further contain a protein other than the modified fibroin.

Examples of the solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and aqueous solutions containing urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride, lithium thiocyanate, etc. These solvents may be used alone or in combination of two or more.

The content of modified fibroin in the dope solution may be 15% by mass or more, 30% by mass or more, 40% by mass or more, or 50% by mass or more, based on the total mass of the dope solution. From the viewpoint of the production efficiency of the dope solution, the content of modified fibroin may be 70% by mass or less, 65% by mass or less, or 60% by mass or less, based on the total mass of the dope solution.

Examples of dissolution promoters include inorganic salts consisting of Lewis acids and Lewis bases as shown below. Examples of Lewis bases include oxoacid ions (nitrate ions, perchlorate ions, etc.), metal oxoacid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, etc. Examples of Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, complex ions, etc. Specific examples of inorganic salts composed of Lewis acids and Lewis bases include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate; calcium salts such as calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium perchlorate, and calcium thiocyanate; iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron thiocyanate; aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate; potassium salts such as potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate; sodium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate; zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate, and zinc thiocyanate; magnesium salts such as magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium thiocyanate; barium salts such as barium chloride, barium bromide, barium iodide, barium nitrate, barium perchlorate, and barium thiocyanate; and strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate.

The content of the solubility enhancer may be 1.0 parts by mass or more, 5.0 parts by mass or more, 9.0 parts by mass or more, 15 parts by mass or more, or 20.0 parts by mass or more, relative to 100 parts by mass of the total amount of the modified fibroin. The content of the solubility enhancer may be 40 parts by mass or less, 35 parts by mass or less, or 30 parts by mass or less, relative to 100 parts by mass of the total amount of the modified fibroin.

When producing the dope solution according to this embodiment, it may be heated to 30 to 90°C. The temperature at which the solution can be dissolved may be appropriately set depending on the solvent used, the type of modified fibroin, etc. The solution may be shaken or stirred to promote dissolution.

The viscosity of the dope solution according to this embodiment may be set appropriately depending on the application of the dope solution. For example, when the dope solution according to this embodiment is used as a spinning stock solution, its viscosity may be set appropriately depending on the spinning method, for example, 100 to 15,000 cP (centipoise) at 35°C, 100 to 30,000 cP (centipoise) at 40°C, etc. The viscosity of the spinning stock solution can be measured using, for example, an "EMS Viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd.

(Protein fiber) The artificial modified fibroin composition of this embodiment may be in the form of a protein fiber. The protein fiber can be obtained, for example, by spinning the above-mentioned dope solution (spinning solution) using a method commonly used for spinning fibroin.

The spinning method is not particularly limited as long as it can spin the modified fibroin of the present invention, and examples of the method include dry spinning, melt spinning, wet spinning, etc. A preferred spinning method is wet spinning.

In wet spinning, the dope solution is extruded from a spinneret (nozzle) into a coagulation liquid (coagulation liquid tank) and the modified fibroin is solidified in the coagulation liquid to obtain an undrawn thread in the shape of a thread. The coagulation liquid may be any solution capable of removing the solvent, such as lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, and 2-propanol, and acetone. Water may be added to the coagulation liquid as appropriate. The temperature of the coagulation liquid is preferably 0 to 30°C. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the spinneret, the extrusion speed is preferably 0.2 to 6.0 ml/hour per hole, and more preferably 1.4 to 4.0 ml/hour. The length of the coagulation liquid tank may be such that the solvent can be removed efficiently, and is, for example, 200 to 500 mm. The take-up speed of the undrawn thread may be, for example, 1 to 20 m/min, and is preferably 1 to 3 m/min. The residence time may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes. Also, drawing (pre-drawing) may be performed in the coagulation liquid. In order to suppress evaporation of the lower alcohol, the coagulation liquid may be kept at a low temperature, and the yarn may be taken up in an undrawn state. The coagulation liquid tank may be provided in multiple stages, and drawing may be performed in each stage or in a specific stage, as necessary.

The undrawn yarn (or pre-drawn yarn) obtained by the above method can be made into a drawn yarn through a drawing process. Examples of drawing methods include wet heat drawing and dry heat drawing.

Wet heat drawing can be carried out in warm water, in a solution of warm water with an organic solvent added, or under steam heating. The temperature may be, for example, 50 to 90°C, with 75 to 85°C being preferred. In wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, with 2 to 8 times being preferred.

Dry heat drawing can be carried out using an electric tubular furnace, a dry heat plate, etc. The temperature may be, for example, 140°C to 270°C, with 160°C to 230°C being preferred. In dry heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, with 1 to 4 times being preferred.

The wet heat stretching and the dry heat stretching may be performed alone, or may be performed in multiple stages or in combination. That is, the first stage stretching may be performed by wet heat stretching and the second stage stretching by dry heat stretching, or the first stage stretching may be performed by wet heat stretching, the second stage stretching may be performed by wet heat stretching, and the third stage stretching may be performed by dry heat stretching, etc.

The final draw ratio in the drawing process is, for example, 5 to 20 times, and preferably 6 to 11 times, relative to the undrawn yarn (or pre-drawn yarn).

After stretching, the protein fibers may be chemically crosslinked between polypeptide molecules in the protein fibers. Examples of functional groups that can be crosslinked include amino groups, carboxyl groups, thiol groups, and hydroxyl groups. For example, the amino group of a lysine side chain contained in a polypeptide can be crosslinked with a carboxyl group of a glutamic acid or aspartic acid side chain through an amide bond by dehydration condensation. Crosslinking may be performed by carrying out a dehydration condensation reaction under vacuum heating, or may be performed using a dehydration condensation agent such as carbodiimide.

Crosslinking between polypeptide molecules may be performed using a crosslinking agent such as carbodiimide or glutaraldehyde, or an enzyme such as transglutaminase. Carbodiimides are compounds represented by the general formula R1N=C=NR2 (wherein R1 and R2 each independently represent an organic group containing an alkyl group or a cycloalkyl group having 1 to 6 carbon atoms). Specific examples of carbodiimides include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diisopropylcarbodiimide (DIC), and the like. Among these, EDC and DIC are preferred because they have a high ability to form amide bonds between polypeptide molecules and are easily crosslinked.

The crosslinking treatment is preferably carried out by applying a crosslinking agent to the protein fibers and crosslinking them by vacuum heating and drying. The crosslinking agent may be applied to the protein fibers in its pure form, or may be applied to the protein fibers after diluting it with a lower alcohol having 1 to 5 carbon atoms, a buffer solution, or the like to a concentration of 0.005 to 10% by mass. The crosslinking treatment is preferably carried out at a temperature of 20 to 45°C for 3 to 42 hours. The crosslinking treatment can impart even higher stress (strength) to the protein fibers.

(Film) The artificially modified fibroin composition according to this embodiment may be in the form of a film. The film can be obtained, for example, by casting the above-mentioned dope liquid onto a substrate surface, followed by drying and/or desolvation.

The viscosity of the dope solution is preferably 15 to 80 cP (centipoise), and more preferably 20 to 70 cP.

When the dope solution is 100% by mass, the concentration of the modified fibroin of the present invention is preferably 3 to 50% by mass, more preferably 3.5 to 35% by mass, and even more preferably 4.2 to 15.8% by mass.

When preparing the dope solution, it may be heated to 30-60°C. It may also be shaken or stirred to promote dissolution.

The substrate may be a resin substrate, a glass substrate, a metal substrate, etc. The substrate is preferably a resin substrate from the viewpoint of easily peeling off the film after cast molding. The resin substrate may be, for example, a polyethylene terephthalate (PET) film, a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a release film having a silicone compound fixed to the surface of these films. The substrate is more preferably a PET film or a release film having a silicone compound fixed to the surface of a PET film, from the viewpoint of being stable against HFIP, DMSO solvent, etc., allowing the dope solution to be stably cast molded, and allowing the film after molding to be easily peeled off.

To explain the specific procedure, first the dope liquid is cast onto the surface of the substrate, and then a film thickness control means such as an applicator, knife coater, or bar coater is used to create a wet film of a predetermined thickness (e.g., 1 to 1,000 μm after drying and/or desolvation).

Drying and/or desolvation can be carried out by a dry or wet method. Examples of dry methods include vacuum drying, hot air drying, and air drying. Examples of wet methods include a method in which the cast film is immersed in a desolvation liquid (also called a coagulation liquid) to remove the solvent. Examples of desolvation liquids include water, alcohol liquids such as lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and mixtures of water and alcohol. The temperature of the desolvation liquid (coagulation liquid) is preferably 0 to 90°C.

The unstretched film after drying and/or desolvation can be uniaxially or biaxially stretched in water. Biaxial stretching may be sequential or simultaneous biaxial stretching. Multi-stage stretching of two or more stages may be performed. The stretching ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times, both longitudinal and transverse. Within this range, it is easy to balance the stress-strain. Underwater stretching is preferably performed at a water temperature of 20 to 90°C. After stretching, the film is preferably heat-set with dry heat at 50 to 200°C for 5 to 600 seconds. This heat setting provides dimensional stability at room temperature. A uniaxially stretched film becomes a uniaxially oriented film, and a biaxially stretched film becomes a biaxially oriented film.

[Method for producing an artificial modified fibroin composition] The artificial modified fibroin composition of the present invention can be produced by a method including a step of preparing the modified fibroin of the present invention. The method for producing an artificial modified fibroin composition of the present invention may further include a step of preparing a modified fibroin solution (e.g., a dope solution) containing the modified fibroin of the present invention.

[Method for improving thermal stability of modified fibroin] The method for improving the thermal stability of modified fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif according to the present embodiment includes increasing the number of consecutive alanine residues in at least one (A) _n motif by inserting one or more alanine residues into at least one (A) _n motif or REP in the fibroin before modification, or by substituting another amino acid residue adjacent to the alanine residue with alanine. Compared with the fibroin before modification, the thermal decomposition temperature (Td) of the modified fibroin is 5°C or higher. The fibroin before modification includes naturally occurring fibroin and modified fibroin.

Here, the (A) _n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 4 to 27. The number of amino acid residues in the (A) _n motif may be 4 to 20, 8 to 20, 10 to 20, 4 to 16, 5 to 10, 6 to 12, 6 to 18, 7 to 10, 7 to 14, 8 to 16, or 11 to 16. The ratio of the number of alanine residues to the total number of amino acid residues in the (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The (A) _n motifs present in a plurality of instances may be the same amino acid sequence as each other, or may be different amino acid sequences. The REPs present in a plurality of instances may be the same amino acid sequence as each other, or may be different amino acid sequences.

The (A) _n motif may have a number of alanine residues of 80% or more, preferably 85% or more, more preferably ₉₀ % or more, even more preferably 95% or more, and even more preferably 100% (meaning composed only of alanine residues). Of the (A) _n motifs present in a plurality in the domain sequence, at least seven are preferably composed only of alanine residues. "Composed only of alanine residues" means that the (A) _n motif has an amino acid sequence represented by (Ala) _k (Ala represents an alanine residue, and k represents an integer of 4 to 27, preferably an integer of 4 to 20, more preferably an integer of 4 to 16).

The method according to the present embodiment includes increasing the number of alanine residues in at least one (A) _n motif in the fibroin before modification. The method according to the present embodiment may increase the number of alanine residues in at least two or more (A) _n motifs in the fibroin before modification, may increase the number of alanine residues in at least five or more (A) _n motifs in the fibroin before modification, may increase the number of alanine residues in at least six or more (A) _n motifs in the fibroin before modification, may increase the number of alanine residues in at least seven or more (A) _n motifs in the fibroin before modification, or may increase the number of alanine residues in all (A) _n motifs in the fibroin before modification. This can improve the thermal stability of the modified fibroin. Increasing the number of consecutive alanine residues in at least one (A) _n motif may be achieved by inserting one or more alanine residues into at least one (A) _n motif or REP in the unmodified fibroin and/or by substituting another amino acid residue adjacent to the alanine residue with alanine. The unmodified fibroin may be naturally occurring or modified fibroin.

The method according to the present embodiment may include, in addition to the modification corresponding to inserting one or more alanine residues into at least one (A) _n motif or REP in the unmodified fibroin, or substituting other amino acid residues adjacent to the alanine residue with alanine, further modifying the amino acid sequence corresponding to substituting, deleting, inserting and/or adding one or more amino acid residues. Substitution, deletion, insertion and/or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial directed mutagenesis. Specifically, it can be performed according to the methods described in literature, such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

The method according to the present embodiment can include, for example, adding a mutation to the gene sequence of the cloned naturally-occurring fibroin, which corresponds to inserting one or more alanine residues into the (A) _n motif or REP, or substituting other amino acid residues adjacent to the alanine residue with alanine. The modified fibroin can also be obtained by, for example, designing an amino acid sequence corresponding to inserting one or more alanine residues into the (A) _n motif or REP, or substituting other amino acid residues adjacent to the alanine residue with alanine, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

In the modified fibroin according to the method of the present embodiment, the total number of alanine residues in the (A) _n motif, which are consecutive for 6 or more residues, is preferably 20% or more of the total number of amino acid residues. The total number of alanine residues in the (A) _n motif, which are consecutive for 6 or more residues, means the total number of alanine residues when only the number of alanine residues in the (A) n motif is counted. Therefore, the number of alanine residues in the (A) n motif, which are consecutive for 5 residues, is not counted. In addition, in the (A) _n motif, when there are no alanine residues in the (A) n motif, due to the presence of other amino acid residues between the alanine residues, the number of alanine residues is not counted.

The total number of six or more consecutive alanine residues in the (A) _n motif may be, for example, 20% or more, 21% or more, 22% _or more, or 23% or more of the total number of all amino acid residues.
It may be 1% or more, 22% or more, or 23% or more.

The fibroin after modification in the method according to this embodiment preferably has a total number of amino acid residues of 580 or more. The total number of amino acid residues may be, for example, 580 or more, 590 or more, 600 or more, 610 or more, or 620 or more.

In the fibroin modified by the method of this embodiment, the hydrophobicity of the REP is preferably -1.0 or more, more preferably -0.9 or more, even more preferably -0.8 or more, even more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.2 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of the REP, and it may be, for example, 1.0 or less, or 0.7 or less. The hydrophobicity of the REP may be, for example, -1.0 or more and 1.0 or less, or 0 or more and 0.7 or less.

In the method according to this embodiment, the thermal decomposition temperature (Td) of the modified fibroin may be at least 5°C higher than that of the fibroin before modification, for example, at least 8°C, at least 10°C, or at least 12°C higher. The fibroin before modification is preferably naturally derived fibroin, more preferably fibroin derived from insects or spiders, and even more preferably large ampullate spider protein (MaSp) or small ampullate spider protein (MiSp) of spiders.

Furthermore, in the method of this embodiment, the glass transition point (Tg) of the modified fibroin may be higher than that of the fibroin before modification, for example, by 5°C or more, 8°C or more, 10°C or more, or 12°C or more.

The modification of the fibroin protein in the method according to this embodiment can be carried out in a manner that results in the preferred aspects described as embodiments of the modified fibroin.

[Products] The modified fibroin or artificial modified fibroin composition of the present invention can be used as fibers (long fibers, short fibers, multifilaments, monofilaments, etc.) or yarns (spun yarns, twisted yarns, false twisted yarns, textured yarns, blended yarns, blended yarns, etc.) in woven fabrics, knitted fabrics, braids, nonwoven fabrics, etc. It can also be used in high-strength applications such as ropes, surgical sutures, flexible fasteners for electrical components, and even bioactive materials for transplants (e.g., artificial ligaments and aortic bands).

In addition, the modified fibroin or artificial modified fibroin composition of the present invention can be applied not only to fibers and films, but also to foams, granules (spherical or non-spherical, etc.), nanofibrils, gels (hydrogels, etc.), resins and their equivalents, which can be produced in accordance with the methods described in JP 2009-505668 A, Japanese Patent No. 5,678,283 A, Japanese Patent No. 4,638,735 A, etc.

The product of this embodiment comprises the modified fibroin of the present invention and is selected from the group consisting of fibers, threads, films, foams, granules, nanofibrils, gels and resins.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

[(1) Synthesis of nucleic acid encoding modified fibroin and construction of expression vector] Based on the base sequence and amino acid sequence of naturally occurring fibroin from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), fibroin and modified fibroin having the amino acid sequences shown in SEQ ID NOs: 6 to 12 were designed.

The amino acid sequence shown in SEQ ID NO:9 (PRT380) is obtained by deleting the consecutive alanine residues in each (A) _n motif of the naturally-occurring fibroin so that it is five, replacing all GGX in REP with GQX, and adding the amino acid sequence shown in SEQ ID NO:4 (tag sequence and hinge sequence) to the N-terminus.

The amino acid sequence shown in SEQ ID NO:10 (PRT525) is obtained by deleting every third (A) _n motif ((A) ₅ ) from the N-terminus to the C-terminus of the naturally occurring fibroin, inserting one [(A) _n motif-REP] just before the C-terminus sequence, replacing all GGX in REP with GQX, inserting two alanine residues on the C-terminus of each (A) _n motif, replacing some glutamine (Q) residues with serine (S) residues, deleting some amino acids on the C-terminus so that the molecular weight is approximately the same as that of PRT380, and adding the amino acid sequence shown in SEQ ID NO:4 (tag sequence and hinge sequence) to the N-terminus.

The amino acid sequence shown in SEQ ID NO:11 (PRT1068) is based on the amino acid sequence shown in SEQ ID NO:12 (PRT564) in which one alanine residue is deleted from the C-terminus of each (A) _n motif and Q in REP is replaced with VFI.

The amino acid sequence shown in SEQ ID NO:6 (PRT1069) is obtained by inserting one alanine residue on the C-terminal side of each (A) _n motif of PRT1068.

The amino acid sequence shown in SEQ ID NO:7 (PRT1070) has two alanine residues inserted on the C-terminal side of each (A) _n motif of PRT1068.

Nucleic acids encoding proteins having the designed amino acid sequences shown in SEQ ID NOs: 6 to 12 were each synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. These five types of nucleic acids were cloned into a cloning vector (pUC118). The nucleic acids were then excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Protein Expression E. coli BLR (DE3) was transformed with a pET22b(+) expression vector containing a nucleic acid encoding a protein having an amino acid sequence shown in SEQ ID NO:6 to 12. 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 containing ampicillin (Table 5) so that the OD ₆₀₀ became 0.005. The culture solution temperature was kept at 30° C., and flask culture was continued until the OD ₆₀₀ became 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 6) so that the OD ₆₀₀ was 0.05, and the transformed E. coli was inoculated. The culture temperature was kept at 37° C., and the pH was controlled to a constant value of 6.9 during culture. The dissolved oxygen concentration in the culture 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 culture temperature was kept at 37°C and the culture was controlled to a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein. 20 hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the target protein was confirmed by the appearance of a band of the target protein size depending on the addition of IPTG.

[(3) Protein purification] (1) Approximately 50 g of E. coli cells expressing the modified fibroin protein and 300 ml of buffer AI (20 mM Tris-HCl, pH 7.4) were added to a centrifuge tube (1,000 ml). The cells were dispersed using a mixer (IKA "T18 Basic Ultra Turrax", level 2), and then centrifuged (11,000 g, 10 minutes, room temperature) using a centrifuge (Kubota "Model 7000"), and the supernatant was discarded.

(2) 300 ml of Buffer AI and 3 ml of 0.1 M PMSF (dissolved in isopropanol) were added to the precipitate (bacterial cells) obtained by centrifugation, and the mixture was dispersed for 3 minutes using the IKA mixer (level 2). The bacterial cells were then repeatedly disrupted three times using a high-pressure homogenizer (GEA Niro Saovi's "Panda Plus 2000").

(3) 300 mL of buffer B (50 mM Tris-HCL, 100 mM NaCl, pH 7.0) containing 3 w/v% SDS was added to the disrupted cells, and the mixture was thoroughly dispersed using the IKA mixer (level 2) and then stirred for 60 minutes using a shaker (Taitec, 200 rpm, 37°C). The mixture was then centrifuged (11,000 g, 30 minutes, room temperature) using the Kubota centrifuge, the supernatant was discarded, and SDS-washed granules (precipitate) were obtained.

(4) The SDS-washed granules were suspended in a DMSO solution containing 1 M lithium chloride to a concentration of 100 mg/mL, and heat-treated at 80° C. for 1 hour. The suspension was then centrifuged (11,000 g, 30 minutes, room temperature) using the Kubota centrifuge, and the supernatant was collected.

(5) Ethanol was prepared in an amount three times that of the collected supernatant, and the collected supernatant was added to the ethanol and allowed to stand at room temperature for 1 hour. The collected protein was then centrifuged (11,000 g, 30 minutes, room temperature) using the Kubota centrifuge to collect the aggregated protein. The aggregated protein was then washed with pure water and collected by centrifugation. This process was repeated three times, after which the moisture was removed using a freeze-dryer and the freeze-dried powder was collected.

[(4) Measurement of thermal decomposition temperature (Td) and glass transition point (Tg)] Measurements were performed using a Shimadzu simultaneous differential thermal and thermogravimetric analyzer (DTG-60H) according to the manual provided by the manufacturer. 10 mg of protein powder was used for each sample, and 20 mg of alumina powder was used as the reference material. The reference material and sample were placed in separate aluminum containers and covered with an aluminum cover. They were heated to a target temperature of 300°C at 10°C/min in a nitrogen atmosphere. Thermal analysis measurement graphs were analyzed using Softwere TA60 (Shimadzu).

The thermal decomposition temperature (Td) was measured by tangent intersection analysis of the differential thermal analysis (DTA) curve. The glass transition temperature (Tg) was measured by tangent intersection analysis of the DTA curve.

[(5) Results] The characteristics of the amino acid sequences of PRT380 and PRT525 are shown in Table 7. The results of comparing the thermal decomposition temperature and glass transition point of PRT380 and PRT525 are shown in Figures 2 and 3, respectively. Compared to PRT380, PRT525 showed a higher thermal decomposition temperature and glass transition point.

The characteristics of the amino acid sequences of PRT1068, PRT1069, and PRT1070 are shown in Table 8. The results of comparing the thermal decomposition temperatures of PRT1068, PRT1069, and PRT1070 are shown in Figure 4. There was a tendency for the thermal decomposition temperature to increase by increasing the number of consecutive alanine residues in each (A) _n motif.

The thermal decomposition temperatures (Td) of PRT525 (SEQ ID NO: 10) and PRT1070 (SEQ ID NO: 7), in which the number of consecutive alanine residues in each (A) _n motif is 7, are compared in Figure 5. The hydrophobicity of the REP of PRT525 is -1.32, and the hydrophobicity of the REP of PRT1070 is 0.21.

PRT1070, which has a higher hydrophobicity of REP, showed a higher thermal decomposition temperature than PRT525.

Claims (13)

A modified fibroin comprising a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m -(A) _n motif,
(A) the total number of consecutive alanine residues in _{the n} motif is 22% or more of the total number of all amino acid residues;
The hydrophobicity of the REP is 0.2 or more;
A modified fibroin having a total number of amino acid residues of 620 or more. [In formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may have the same amino acid sequence as each other or different amino acid sequences.] The amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3,
(A) A modified fibroin, in which the total number of consecutive alanine residues in _n motifs is 20% or more of the total number of amino acid residues. The modified fibroin according to claim 1 or 2, further comprising a tag sequence at either or both of the N-terminus and C-terminus. The modified fibroin according to claim 3, wherein the tag sequence comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5. The amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8,
(A) A modified fibroin, in which the total number of consecutive alanine residues in _n motifs is 20% or more of the total number of amino acid residues. The modified fibroin according to any one of claims 1 to 5, having a thermal decomposition temperature (Td) of 268°C or higher. A nucleic acid encoding the modified fibroin according to any one of claims 1 to 6. A method for producing modified fibroin, comprising the steps of:
expressing the nucleic acid by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence;
A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin according to any one of claims 1 to 6 . A method for improving the thermal stability of a modified fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m -(A) _n motif, comprising:
The method includes increasing the number of consecutive alanine residues in at least one (A) _n motif by inserting one or more alanine residues into at least one (A) _n motif or REP in the unmodified fibroin or by substituting another amino acid residue adjacent to the alanine residue with alanine;
A method in which the thermal decomposition temperature (Td) of the modified fibroin is 5°C or higher compared to that of the unmodified fibroin. [In formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in the (A) _n motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.] The method according to claim 9, wherein the fibroin before modification is naturally occurring fibroin. The method according to claim 10, wherein the naturally occurring fibroin is fibroin from an insect or arachnid. The method according to claim 11, wherein the naturally occurring fibroin is arachnid major ampullate spider protein (MaSp) or minor ampullate spider protein (MiSp). The modified fibroin according to any one of claims 1 to 6 is contained therein,
1. An article of manufacture selected from the group consisting of fibers, yarns, films, foams, granules, nanofibrils, gels and resins.