Patent Application
20190233481
The present invention relates to a modified fibroin. More particularly, the present invention relates to a modified fibroin having an amino acid sequence with a locally high hydropathy index. The present invention also relates to a nucleic acid encoding a modified fibroin, an expression vector including the nucleic acid sequence, a host transformed with the expression vector, and a product made from a modified fibroin.
Fibroin is a type of fibrous protein and contains up to 90% of glycine, alanine and serine residues leading to the formation of a β-pleated sheet (Non-Patent Literature 1). Proteins (silk proteins, hornet silk proteins, and spider silk proteins) and the like constituting the yarn produced by insects and spiders are known as fibroin.
Silk proteins have excellent mechanical properties, hygroscopic properties and deodorizing properties and are widely used as raw materials for garments. In addition, the silk yarn is an immuno-tolerant natural fiber and has high biocompatibility and is therefore also used for surgical sutures.
Up to seven types of silk glands exist in spider, each producing fibroin (spider silk protein) with different properties. According to the organ of the source, spider silk proteins are designated a major ampullate spider protein (MaSp) with high toughness, a minor ampullate spider protein (MiSp) with high elongation, and flagelliform (Flag), tubuliform, aggregate, aciniform, and pyriform spider silk proteins. In particular, structural studies have been intensively conducted in the major ampullate spider protein exhibiting high toughness due to having excellent strength and elongation (Patent Literature 1 and Patent Literature 2).
As a structure specific to fibroin, a structure in which amino acid motifs classified as GPGXX, an extended region rich in alanine residues ((A)n or (GA)n), GGX, and a spacer are repeated is known (Non-Patent Literature 2). In addition, it has been reported that substitution of the (GA)n motif with the (A)n motif leads to decreased elongation but increased tensile strength, an increasing number of GPGXX motifs leads to increased elongation, and substitution of several GPGXX motifs with the (A)n motifs leads to increased tensile strength (Patent Literature 2). In addition, the GGX and GPGXX motifs are thought to have a flexible helical structure that imparts elasticity to yarns (Patent Literature 3).
Recombinant spider silk proteins and recombinant silk proteins are produced in several heterologous protein production systems. For example, transgenic goat, transgenic silkworm, or recombinant plant or mammalian cells are utilized (Non-Patent Literature 3). However, these production systems exhibit a low production rate and are not suitable for mass production meeting the commercial level (Patent Literature 4 and Patent Literature 5). Although many cases of production of recombinant fibroin by organisms such as yeast, mold, gram-negative bacterium and gram-positive bacterium as a production system capable of mass production have also been reported and certain outcomes have been achieved, it has not been possible to achieve industrial mass production of the recombinant fibroin having excellent elongation and tensile strength (Patent Literature 5).
Molded articles obtained by molding spider yarn proteins into various shapes are known. For example, Patent Literature 6 discloses a film using a spider yarn protein. Also, Patent Literature 7 discloses polypeptide particles derived from spider yarn protein.
Due to its excellent properties, fibroin has drawn attention as a new material in various industrial fields such as medicine, aviation, and clothing. Also, due to its applicability to molded articles such as a film, which has been produced from a material derived from petroleum conventionally, fibroin has drawn attention as an alternative material (bioplastic). However, it is necessary to further improve the productivity of fibroin in order to achieve a quantity required for commercial level production.
An object of the present invention is to provide a modified fibroin having improved productivity and having improved bending strength when formed into a molded article.
As a result of extensive studies on methods capable of industrial mass production, the present inventors have found that when modified to a fibroin having an amino acid sequence with a locally high hydropathy index, it is possible to improve productivity and improve the bending strength of a molded article produced with the fibroin. The present invention has been completed on the basis of such novel findings.
That is, the present invention relates to, for example, each of the following inventions.
[1] A modified fibroin, including:
a domain sequence represented by Formula 1: [(A)n motif-REP]m,
in which the domain sequence has an amino acid sequence containing a region with locally high hydropathy index equivalent to an amino acid sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues with a high hydropathy index and/or one or a plurality of amino acid residues with a high hydropathy index are inserted into REP, as compared to naturally occurring fibroin.
[In Formula 1, (A)n motif represents an amino acid sequence consisting of 4 to 20 amino acid residues and the number of alanine residues relative to the total number of amino acid residues in the (A)n motif is 83% 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 or different amino acid sequences, a plurality of REPs may be the same amino acid sequence or different amino acid sequences.]
[2] The modified fibroin according to [1], in which the region with locally high hydropathy index is constituted by two to four consecutive amino acid residues.
[3] The modified fibroin according to [1] or [2], in which the amino acid residues with a high hydropathy index are selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A).
[4] A modified fibroin, including:
a domain sequence represented by Formula 1: [(A), motif-REP]m,
in which in a case where in all REPs included in a sequence excluding a sequence from a (A)n motif located the most C-terminal side to a C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or more is set as x, and the total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as y, x/y is 6.2% or more.
[In Formula 1, the (A)n motif represents an amino acid sequence consisting of 4 to 20 amino acid residues and the number of alanine residues relative to the total number of amino acid residues in the (A)n motif is 83% 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 or different amino acid sequences, and a plurality of REPs may be the same amino acid sequence or different amino acid sequences.]
[5] The modified fibroin according to any one of [1] to [4], in which the fibroin has, in addition to the amino acid sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues with a high hydropathy index and/or one or a plurality of amino acid residues with a high hydropathy index are inserted into REP, an amino acid sequence corresponding to substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues, as compared to naturally occurring fibroin.
[6] The modified fibroin according to [5], in which the naturally occurring fibroin is a fibroin derived from an insect or a spider.
[7] The modified fibroin according to [5], in which the naturally occurring fibroin is a major ampullate spider protein (MaSp) or minor ampullate spider protein (MiSp) of spiders.
[8] A modified fibroin, including:
an amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5, or
an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5.
[9] The modified fibroin according to any one of [1] to [8], further including a tag sequence at either or both of a N-terminal and a C-terminal.
[10] The modified fibroin according to [9], in which the tag sequence includes an amino acid sequence set forth in SEQ ID NO: 6.
[11] A modified fibroin, including:
an amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11, or
an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11.
[12] A nucleic acid that encodes the modified fibroin according to [1] to [11].
[13] A nucleic acid that hybridizes with a complementary strand of the nucleic acid according to [12] under stringent conditions and encodes a modified fibroin including a domain sequence represented by Formula 1: [(A)n motif-REP]m.
[In Formula 1, the (A)n motif represents an amino acid sequence consisting of 4 to 20 amino acid residues and the number of alanine residues relative to the total number of amino acid residues in the (A)n motif is 83% 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 or different amino acid sequences, and a plurality of REPs may be the same amino acid sequence or different amino acid sequences.]
[14] A nucleic acid having 90% or more sequence identity with the nucleic acid according to [13] and encoding a modified fibroin including a domain sequence represented by Formula 1: [(A) motif-REP]m.
[In Formula 1, the (A)n motif represents an amino acid sequence consisting of 4 to 20 amino acid residues and the number of alanine residues relative to the total number of amino acid residues in the (A)n motif is 83% 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 or different amino acid sequences, and a plurality of REPs may be the same amino acid sequence or different amino acid sequences.]
[15] An expression vector, comprising:
the nucleic acid sequence according to any one of [12] to [14]; and
one or a plurality of regulatory sequences operably linked thereto.
[16] The expression vector according to [15], which is a plasmid vector or a viral vector.
[17] A host transformed with the expression vector according to [15] or [16].
[18] The host according to [17], which is a prokaryote.
[19] The host according to [18], wherein the prokaryote is a microorganism belonging to a genus selected from the group consisting of Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
[20] The host according to [17], which is a eukaryote.
[21] The host according to [20], in which the eukaryote is a yeast, a filamentous fungus or an insect cell.
[22] The host according to [21], in which the yeast is a yeast belonging to a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia and Hansenula.
[23] The host according to [22], in which the yeast belonging to the genus Saccharomyces is Saccharomyces cerevisiae, the yeast belonging to the genus Schizosaccharomyces is Schizosaccharomyces pombe, and the yeast belonging to the genus Kluyveromyces is Kluyveromyces lactis, the yeast belonging to the genus Trichosporon is Trichosporon pullulans, the yeast belonging to the genus Schwaniomyces is Schwanniomyces alluvius, the yeast belonging to the genus Pichia is Pichia pastoris, the yeast belonging to the genus Candida is Candida albicans, the yeast belonging to the genus Yarmwia is Yarrowia lipolytica, and the yeast belonging to the genus Hansenula is Hansenula polymorpha.
[24] The host according to [21], in which the filamentous fungus is a filamentous fungus belonging to a genus selected from the group consisting of Aspergillus, Penicillium and Mucor.
[25] The host according to [24], in which the filamentous fungus belonging to the genus Aspergillus is Aspergillus oryzae, the filamentous fungus belonging to the genus Penicillium is Penicillium chrysogenum, and the filamentous fungus belonging to the genus Mucor is Mucor fragilis.
[26] The host according to [21], in which the insect cell is a lepidopteran insect cell.
[27] The host according to [26], in which the insect cell is an insect cell derived from Spodoptera frugiperda or an insect cell derived from Trichoplusia ni.
[28] A product including the modified fibroin according to any one of [1] to [11] and selected from the group consisting of a fiber, a yarn, a filament, a film, a foam, a sphere, a nanofibril, a hydrogel, a resin and an equivalent thereof.
According to the present invention, it is possible to provide a modified fibroin having improved productivity and having improved bending strength when formed into a molded article.
Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
[Modified Fibroin]
The modified fibroin according to the present invention is a protein including a domain sequence represented by Formula 1: [(A)n motif-REP]m. In the modified fibroin, an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence, although not limited thereto, are typically regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of amino acids of about 100 residues.
The term “modified fibroin” as used herein means a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin. The term “naturally occurring fibroin” as used herein is also a protein including a domain sequence represented by Formula 1: [(A)n motif-REP]m.
The “modified fibroin” may be a fibroin whose amino acid sequence has been modified based on naturally occurring fibroin (for example, a fibroin whose amino acid sequence has been modified by altering a gene sequence of cloned naturally occurring fibroin) or a fibroin artificially designed and synthesized independently of naturally occurring fibroin (for example, a fibroin having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence), as long as it has the amino acid sequence specified in the present invention.
The term “domain sequence” as used herein refers to an amino acid sequence which produces a crystalline region (typically, corresponds to (A)n motif of an amino acid sequence) and an amorphous region (typically, corresponds to REP of an amino acid sequence) peculiar to fibroin and means an amino acid sequence represented by Formula 1: [(A)n motif-REP]m. Here, the (A)n motif represents an amino acid sequence consisting of 4 to 20 amino acid residues and the number of alanine residues relative to the total number of amino acid residues in the (A)n motif is 83% or more. The 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 (AX) motifs may be the same amino acid sequence or different amino acid sequences. A plurality of REPs may be the same amino acid sequence or different amino acid sequences.
The (A)n motif may be such that the number of alanine residues is 83% or more relative to the total number of amino acid residues in the (A)n motif, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even still more preferably 100% (which means that the (A)n motif consists of only alanine residues). It is preferred that at least seven of a plurality of (A)n motifs in the domain sequence consist of only alanine residues. The phrase “consist of only alanine residues” means that the (A)n motif has an amino acid sequence represented by (A)n (where A represents an alanine residue and n represents an integer of 4 to 20 and preferably an integer of 4 to 16). It is preferable that at least one motif selected from GGX and GPGXX (here, X represents an amino acid residue other than a glycine residue) is contained in the amino acid sequence of REP. With REP containing these motifs, the elongation of fibroin is allowed to be improved.
A modified fibroin according to an embodiment of the present invention may include a domain sequence having an amino acid sequence locally containing a region with a high hydropathy index equivalent to an amino acid sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues with a high hydropathy index and/or one or a plurality of amino acid residues with a high hydropathy index are inserted into REP, as compared to naturally occurring fibroin.
It is preferable that in the modified fibroin according to the present embodiment, the region with locally high hydropathy index is constituted by two to four consecutive amino acid residues. With this configuration, the effect of the present invention is more significantly exhibited.
It is more preferable that in the modified fibroin according to the present embodiment, the amino acid residues with a high hydropathy index are selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A). With this configuration, the effect of the present invention of improvement of productivity of the fibroin in the recombinant protein production system and remarkable improvement of bending strength of the molded article produced with the fibroin can be more stably exerted.
The modified fibroin according to the present embodiment may further have modifications of an amino acid sequence corresponding to substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues as compared to naturally occurring fibroin, in addition to the amino acid sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues with a high hydropathy index and/or one or a plurality of amino acid residues with a high hydropathy index are inserted into REP, as compared to naturally occurring fibroin.
The modified fibroin according to the present embodiment may be obtained by, with respect to a gene sequence of cloned naturally occurring fibroin, substituting one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydropathy index) with a hydropathy amino acid residues (for example, amino acid residues having a positive hydropathy index), and/or inserting one or a plurality of hydropathy amino acid residues into REP. Further, for example, the modified fibroin according to the present embodiment may also be obtained by designing an amino acid sequence equivalent to an amino acid sequence in which with respect to the amino acid sequence of naturally occurring fibroin, one or a plurality of hydrophilic amino acid residues in REP are substituted with hydropathy amino acid residues and/or one or a plurality of hydropathy amino acid residues are inserted into REP, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, with respect to the amino acid sequence naturally occurring fibroin, in addition to the modification corresponding to substitution of one or a plurality of hydrophilic amino acid residues in REP with hydropathy amino acid residues and/or insertion of one or a plurality of hydropathy amino acid residues into REP, further modification of amino acid sequence corresponding to substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues may be carried out. The substitution, deletion, insertion and/or addition of amino acid residues may be carried out by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, the modifications may be carried out by a method described in literature such as Nucleic Acid Res. 10, 6487 (1982), and Methods in Enzymology, 100, 448 (1983).
Naturally occurring fibroin is a protein including a domain sequence represented by Formula 1: [(A)n motif-REP]m, specifically, for example, a fibroin produced by insects or spiders.
Examples of the fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligurajaponica, Antheraea mylitta, and Antheraea assama; and hornet silk proteins discharged by larvae of Vespa simillima xanthoptera.
A more specific example of the fibroin produced by insects may be a silkworm fibroin L chain (GenBank Accession No. M76430 (nucleotide sequence), AAA27840.1 (amino acid sequence)).
Examples of the fibroin produced by spiders include spider silk proteins produced by spiders belonging to the genus Araneus such as Araneus ventricosus, Araneus diadematus, Araneus pinguis, Araneus pentagrammicus and Araneus nojimai, spiders belonging to the genus Neoscona such as Neoscona scylla, Neoscona nautica, Neoscona adianta and Neoscona scylloides, spiders belonging to the genus Pronus such as Pronous minutes, spiders belonging to the genus Cyrtarachne such as Cyrtarachne bufo and Cyrtarachne inaequalis, spiders belonging to the genus Gasteracantha such as Gasteracantha kuhli and Gasteracantha mammosa, spiders belonging to the genus Ordgarius such as Ordgarius hobsoni and Ordgarius sexspinosus, spiders belonging to the genus Argiope such as Argiope amoena, Argiope minuta and Argiope bruennich, spiders belonging to the genus Arachnura such as Arachnura logio, spiders belonging to the genus Acusilas such as Acusilas coccineus, spiders belonging to the genus Cytophora such as Cyrtophora moluccensis, Cyrtophora exanthematica and Cyrtophora unicolor, spiders belonging to the genus Poltys such as Poltys illepidus, spiders belonging to the genus Cyclosa such as Cyclosa octotuberculata, Cyclosa sedeculata, Cyclosa vallata and Cyclosa atrata, and spiders belonging to the genus Chorizopes such as Chorizopes nipponicus; and spider silk proteins produced by spiders belonging to the genus Tetragnatha such as Tetragnatha praedonia, Tetragnatha maxillosa, Tetragnatha extensa and Tetragnatha squamata, spiders belonging to the genus Leucauge such as Leucauge magnifica, Leucauge blanda and Leucauge subblanda, spiders belonging to the genus Nephila such as Nephila clavata and Nephila pilipes, spiders belonging to the genus Menosira such as Menosira ornata, spiders belonging to the genus Dyschiriognatha such as Dyschiriognatha tenera, spiders belonging to the genus Latrodectus such as Latrodectus mactans, Latrodectus hasseltii, Latrodectus geometricus and Latrodectus tredecimguttatus, and spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include traction fiber proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
More specific examples of the fibroin produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank Accession Number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession Number AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession Number AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major angullate spidroin 1 [derived from Latmdectus hesperus] (GenBank Accession Number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank Accession Number AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major anpullate spidroin 1 [derived from Euprosthenops australis](GenBank Accession Number CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)) and major ampullate spidroin 2 [Euprosthenops 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)), and minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank Accession Number ABR37278.1 (amino acid sequence)).
As a further specified example of naturally occurring fibroin, fibroin whose sequence information is registered in NCBI GenBank may be mentioned. For example, sequences thereof may be confirmed by extracting sequences in which spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION among sequences containing INV as DIVISION among sequence information registered in NCBI GenBank, sequences in which a specific character string of products is described from CDS, or sequences in which a specific character string is described from SOURCE to TISSUE TYPE.
A modified fibroin according to another embodiment includes a domain sequence represented by Formula 1: [(A)n motif-REP]m and has an amino acid sequence in which x/y is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from a (A)n motif located to most C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or more is set as x, and the total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as y.
Regarding the hydropathy index of amino acid residues, known indices from (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. 105-132) may be used as a reference. Specifically, the hydropathy index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table I below.
The calculation method of x/y will be described in more detail. In the calculation, the sequence excluding the sequence from the (A), motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence (hereinafter also referred to as “sequence A”) is used. First, in all REPs included in the sequence A, average values of hydropathy indices of four consecutive amino acid residues are calculated. The average value of the hydropathy indices is obtained by dividing the sum total of HI of each of the amino acid residues contained in the four consecutive amino acid residues by 4 (number of amino acid residues). The average value of the hydropathy indices is obtained for all of the four consecutive amino acid residues (each of the amino acid residues is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydropathy indices of the four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue is determined to be included in the “four consecutive amino acid residues having an average value of the hydropathy indices of 2.6 or more” multiple times, the amino acid residue is counted as one amino acid residue in the region. The total number of amino acid residues included in the region is set as x. The total number of amino acid residues included in the sequence A is set as y.
For example, in a case where the feature “four consecutive amino acid residues whose average value of the hydropathy indices is 2.6 or more” is extracted from 20 places (no overlap), in the region where the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more, the number of the four consecutive amino acid residues (no overlap) is 20, and thus x is 20×4=80. In addition, for example, when two of the “four consecutive amino acid residues having an average value of the hydropathy indices of 2.6 or more” overlap by one amino acid residue, in the region where the average value of the hydropathy indices of the four consecutive amino acid residues is 2.6 or more, the number of amino acid residues being included is 7(x=2×4−1=7. “−1” is the deduction of overlap). For example, in the case of the domain sequence shown in
Here, x/y in naturally occurring fibroin will be described. First, as described above, 663 types of fibroins (415 types of fibroins derived from spiders among them) were extracted by confirming fibroins with amino acid sequence information registered in NCBI GenBank by the method exemplified. Among all the extracted fibroins, x/y was calculated by the above-mentioned calculation method with respect to the naturally occurring fibroins including the domain sequence represented by Formula 1: [(A)n motif-REP]m. The results are shown in
In the modified fibroin according to the present embodiment, x/y is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, even still more preferably 20% or more, and still further preferably 30% or more. The upper limit of x/y is not particularly limited, but it may be 45% or less, for example.
The modified fibroin according to the present embodiment may be obtained by, for example, modifying an amino acid sequence of cloned naturally occurring fibroin into the amino acid sequence locally containing a region with locally high hydropathy index by substituting one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydropathy index) with hydropathy amino acid residues (for example, amino acid residues having a positive hydropathy index), and/or inserting one or a plurality of hydropathy amino acid residues into REP, such that the x/y condition is satisfied. Alternatively, the modified fibroin according to the present embodiment may also be obtained, for example, by designing an amino acid sequence satisfying the x/y condition based on the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to substitution of one or a plurality of amino acid residues in REP with amino acid residues with a high hydropathy index and/or insertion of one or a plurality of amino acid residues with a high hydropathy index into REP, as compared to the amino acid sequence of naturally occurring fibroin, further modification corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may be carried out.
The amino acid residues with a high hydropathy index is preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A), and more preferably valine (V), leucine (L), and isoleucine (I), but not particularly thereto.
A more specific example of the modified fibroin according to the present invention may be a modified fibroin containing (i) an amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5, or (ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5.
The modified fibroin of (i) will be described. The amino acid sequence set forth in SEQ ID NO: 1 is obtained by deleting consecutive alanine residues in the (A)n motif of an amino acid sequence in naturally occurring fibroin such that the number of the consecutive alanine residues becomes five. The amino acid sequence set forth in SEQ ID NO: 2 is obtained by inserting an amino acid sequences consisting of three amino acid residues (VLI) at two sites for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 1, and deleting a part of the amino acid on the N-terminal side therefrom such that the molecular weight thereof is set to be approximately the same as that of the amino acid sequence set forth in SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 3 is obtained by inserting two alanine residues at the C-terminal side of each (A)n motif of the amino acid sequence set forth in SEQ ID NO: 1, and further substituting a part of glutamine (Q) residues with serine (S) residues and deleting a part of amino acids on the N-terminal side therefrom such that the molecular weight thereof becomes approximately the same as that of SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 4 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at one site for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 3. The amino acid sequence set forth in SEQ ID NO: 5 is obtained by inserting an amino acid sequences consisting of three amino acid residues (VLI) at two sites for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 3.
The modified fibroin of (i) may consist of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5.
The modified fibroin of (ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5. The modified fibroin of (ii) is also a protein including a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
The modified fibroin of (ii) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5, and preferably has an amino acid sequence in which x/y is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from a (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or more is set as x, and the total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as y.
The above-mentioned modified fibroin may include a tag sequence at either or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
The tag sequence may be, for example, an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are arranged and has a property of specifically binding to a metal ion such as nickel, so it can be used for isolation of modified fibroin by chelating metal chromatography. A specific example of the tag sequence may be an amino acid sequence set forth in SEQ ID NO: 5 (amino acid sequence including His tag).
In addition, a tag sequence such as glutathione-S-transferase (GST) that specifically binds to glutathione or a maltose binding protein (MBP) that specifically binds to maltose can also be used.
Further, an “epitope tag” utilizing an antigen-antibody reaction can also be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, a myc tag, and a FLAG tag. The modified fibroin can easily be purified with high specificity by utilizing an epitope tag.
It is also possible to use a tag sequence which can be cleaved with a specific protease. By treating a protein adsorbed through the tag sequence with protease, it is also possible to recover the modified fibroin cleaved from the tag sequence.
A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (iii) an amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11, or (iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11.
The amino acid sequences set forth in SEQ ID NOs: 8, 10 and 11 are obtained by adding the amino acid sequence set forth in SEQ ID NO: 6 (including a His tag) to the N-terminals of the amino acid sequences set forth in SEQ ID NOs: 2, 4 and 5, respectively.
The modified fibroin of (iii) may consist of an amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11.
The modified fibroin of (iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11. The modified fibroin of (iv) is also a protein including a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
The modified fibroin of (iv) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11, and preferably has an amino acid sequence in which x/y is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or more is set as x, and the total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as y.
The above-mentioned modified fibroin may include a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
[Nucleic Acid]
The nucleic acid according to the present invention encodes the modified fibroin according to the present invention. Specific examples of the nucleic acid include nucleic acids encoding a modified fibroin including an amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5, or a protein or the like having an amino acid sequence (tag sequence) set forth in SEQ ID NO: 6 attached to either or both of the N-terminal and C-terminal of these amino acid sequences.
A nucleic acid according to an embodiment is a nucleic acid that hybridizes with a complementary strand of the nucleic acid that encodes the modified fibroin according to the present invention under stringent conditions and includes a domain sequence represented by Formula 1: [(A)n motif-REP]m in which x/y is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from a (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average value of hydropathy indices of four consecutive amino acid residues is 2.6 or more is set as x, and the total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is set as y.
The term “stringent conditions” refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. The “stringent conditions” may be any of low stringent conditions, moderately stringent conditions and highly stringent conditions. The low stringent conditions mean that hybridization occurs only in the case where there is at least 85% or more identity between the sequences, and include, for example, conditions of hybridization at 42° C. using 5×SSC containing 0.5% SDS. The moderately stringent conditions mean that hybridization occurs only in the case where there is at least 90% or more identity between the sequences, and include, for example, conditions of hybridization at 50° C. using 5×SSC containing 0.5% SDS. The highly stringent conditions mean that hybridization occurs only in the case where there is at least 95% or more identity between the sequences, and include, for example, conditions of hybridization at 60° C. using 5×SSC containing 0.5% SDS.
[Host and Expression Vector]
An expression vector according to the present invention has a nucleic acid sequence according to the present invention and one or a plurality of regulatory sequences operably linked thereto. The regulatory sequence is a sequence (for example, a promoter, an enhancer, a ribosome binding sequence, or a transcription termination sequence) that controls the expression of a recombinant protein in a host, and can be appropriately selected depending on the type of the host. The type of the expression vector such as a plasmid vector, a viral vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector can be appropriately selected depending on the type of the host.
The host according to the present invention is a host which has been transformed with the expression vector according to the present invention. Both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used as hosts.
As the expression vector, an expression vector which can autonomously replicate in a host cell or can be incorporated into a chromosome of a host and which contains a promoter at a position capable of transcribing the nucleic acid according to the present invention is suitably used.
In the case where a prokaryote such as a bacterium is used as a host, the expression vector according to the present invention is preferably a vector which is capable of autonomous replication in the prokaryote and at the same time includes a promoter, a ribosome binding sequence, a nucleic acid according to the present invention and a transcription termination sequence. A gene that controls a promoter may be included.
Examples of the prokaryote include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
Examples of microorganisms belonging to the genus Escherichia include Echerichia coli BL21 (Novagen, Inc.), Escherichia coli BL21 (DE3) (Life Technologies Corporation), Escherichia coli BLR (DE3) (Merck KGaA), 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), Escherichia coli No. 49, Escherichia coli Rosetta (DE3) (Novagen, Inc.), Escherichia coli TB1, Escherichia coli Tuner (Novagen, Inc.), Escherichia coli Tuner (DE3) (Novagen, Inc.), 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 centrosporus, Brevibacillus formosus, Brevibacillus invocatus, Brevibacillus laterosporus, Brevibacillus limnophilus, Brevibacillus parabrevis, Brevibacillus reuszeri, Brevibacillus thermoruber, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevibacillus brevis 47-5Q (JCM 8975), Brevibacillus choshinensis HPD31 (FERM BP-1087), Brevibacillus choshinensis HPD31-S(FERM BP-6623), Brevibacillus choshinensis HPD31-OK (FERM BP-4573), and Brevibacillus choshinensis SP3 strain (manufactured by Takara Bio, Inc.).
Examples of microorganisms belonging to the genus Serratia include Serratia liquefacience ATCC 14460, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, 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 ammoniaphilum ATCC 15354.
Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC 14067) ATCC 13826, ATCC 14067, Brevibacterium immariophilum ATCC 14068, Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC 13869) ATCC 13665, ATCC 13869, Brevibacterium roseum ATCC 13825, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium tiogenitalis ATCC 19240, Brevibacterium album ATCC 15111, and Brevibacterium cerinum ATCC 15112.
Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC 6871, ATCC 6872, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 14067, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium alkanolyticum ATCC 21511, Corynebacterium callunae ATCC 15991, Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, Corynebacterium lilium ATCC 15990, Corynebacterium melassecola ATCC 17965, Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), and Corynebacterium herculis ATCC 13868.
Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas fulva, and Pseudomonas sp. D-0110.
As a method for introducing an expression vector into the foregoing host cell, any method can be used as long as it introduces DNA into the host cell. Examples thereof include a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], a protoplast method (Japanese Unexamined Patent Publication No. 563-248394), or a method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979).
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 the vector into which the nucleic acid according to the present invention is introduced (hereinafter, simply referred to as “vector”) include pBTrp2, pBTac1, and pBTac2 (all commercially available from Boehringer Mannheim GmbH), pKK233-2 (manufactured by Pharmacia Corporation), pSE280 (manufactured by Invitrogen Corporation), pGEMEX-1 (manufactured by Promega Corporation), pQE-8 (manufactured by QIAGEN Corporation), pKYP10 (Japanese Unexamined Patent Publication No. S58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(−) (manufactured by Stratagene Corporation), pTrs30 [constructed from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [constructed from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [constructed from Escherichia coli IGHA2 (FERM B-400), Japanese Unexamined Patent Publication No. S60-221091], pGKA2 [constructed from Escherichia coli IGKA 2 (FERM BP-6798), Japanese Unexamined Patent Publication No. 60-221091], pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094, 5,160,735), pSupex, pUBll0, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia Corporation), and pET systems (manufactured by Novagen, Inc.).
In the case where Escherichia coli is used as a host, pUC18, pBluescriptl, pSupex, pET22b, pCold, or the like can be mentioned as a suitable vector.
Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110 or pHY500 (Japanese Unexamined Patent Publication No. H2-31682), pNY700 (Japanese Unexamined Patent Publication No. H4-278091), pHY4831 (J. Bacteriol., 1987, 1239-1245), pNU200 (UDAKA Shigezou, Journal of the Agricultural Chemical Society of Japan, 1987, 61: 669-676), pNUl00 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU211R2L5 (Japanese Unexamined Patent Publication No. H7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58: 525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177: 745-749), and pHT210 (Japanese Unexamined Patent Publication No. H6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363), which are known as Bacillus subtilis vectors; and pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569) which is a shuttle vector between Escherichia coli and a microorganism belonging to the genus Brevibacillus.
The promoter is not limited as long as it functions in a host cell. Examples thereof include promoters derived from Escherichia coli or phage such as a trp promoter (Ptrp), a lac promoter, a PL promoter, a PR promoter, and a T7 promoter. Also, promoters artificially designed and modified, such as a promoter (Ptrpx2) in which two Ptrp are connected in series, 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 initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). In the expression vector according to the present invention, a transcription termination sequence is not always necessary for the expression of the nucleic acid according to the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
Examples of eukaryotic hosts include yeast, filamentous fungi (mold and the like), and insect cells.
Examples of the yeast include yeasts belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, Hansemda, and the like. More specific examples of the yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Trichosporon pullulans, Schwanniomyces alluvius, Schwanniomyces occidentalis, Candida utilis, Pichia pastoris, Pichia angusta, Pichia methanolica, Pichia polymorpha, Pichia stipitis, Yarrowia lipolytica, and Hansenula polymorpha.
It is preferred that the expression vector in the case where yeast is used as a host cell usually include an origin of replication (in the case where amplification in a host is required), a selection marker for propagation of the vector in Escherichia coli, a promoter and a terminator for recombinant protein expression in yeast, and a selection marker for yeast.
In the case where the expression vector is a non-integrating vector, it is preferable to further include an autonomously replicating sequence (ARS). This makes it possible to improve the stability of the expression vectors in cells (Myers, A. M., et al. (1986) Gene 45: 299-310).
Examples of the vector in the case where yeast is used as a host include YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), YIp, pHSI9, pHSI5, pA0804, pHIL301, pHIL-S, pPIC9K, pPICZα, pGAPZα, and pPICZ B.
The promoter is not limited as long as it can be expressed in yeast. Examples of the promoter include a promoter of glycolytic genes such as hexose kinase, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a gal 1 promoter, a gal 10 promoter, a heat shock polypeptide promoter, an MFa1 promoter, a CUP 1 promoter, a pGAP promoter, a pGCW14 promoter, an AOX1 promoter, and an MOX promoter.
As a method for introducing an expression vector into yeast, any method can be used as long as it introduces DNA into yeast. Examples thereof include an electroporation method (Methods Enzymol., 194, 182 (1990)), a spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), a lithium acetate method (J. Bacteriol., 153, 163 (1983)), and a method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Examples of filamentous fungi include fungi belonging to the genus Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, Penicillium, Myceliophtora, Botryts, 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 niger, Aspergillus nidulans, Aspergillus parasiticus, Aspergillus frcuum, Aspergillus phoeicus, Aspergillus foetidus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma reseei, Chrysosporium lucknowense, Thermoascus, Sporotrichum, Sporotrichum cellulophilum, Talaromyces, Thielavia terrestris, Thielavia, Neurospora crassa, Fusarium oxysporus, Fusarium graminearum, Fusarium venenatum, Humicola insolens, Penicillium chrysogenum, Penicillium camemberti, Penicillium canescens, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium purpurogenum, Penicillium roqueforti, Myceliophtaora thermophilum, Mucor ambiguus, Mucor circinelloides, Mucor fragilis, Mucor hiemalis, Mucor inaequisporus, Mucor oblongiellipticus, Mucor racemosus, Mucor recurvus, Mocor saturninus, Mocor subtilissmus, Ogataea polymorpha, Phanerochaete chrysosporium, Rhizomucor miehei, Rhizomucor pusillus, and Rhizopus arrhizus.
The promoter in the case where the host is a filamentous fungus may be any one of a gene related to a glycolytic system, a gene related to constitutive expression, an enzyme gene related to hydrolysis, and the like. Specific examples thereof include amyB, glaA, agdA, glaB, TEF1, xynF1 tannase gene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, and catB.
Introduction of the expression vector into filamentous fungi can be carried out by a conventionally known method. Examples thereof include the method of Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)], a protoplast method [Mol. Gen. Genet., 168:111 (1979)], a competent method [J. Mol. Biol., 56: 209 (1971)], and an electroporation method.
Insect cells include, for example, 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.
Examples of the vector in the case where an insect cell is used as a host include baculoviruses such as Autographa californica nuclear polyhedrosis virus which is a virus that infects insects belonging to the family Noctuidae (Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)).
In the case where an insect cell is used as a host, a 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), or Bio/Technology, 6, 47 (1988). That is, a recombinant gene transfer vector and a baculovirus are co-introduced into an insect cell to obtain a recombinant virus (expression vector) in an insect cell culture supernatant, and then the recombinant virus is further infected into an insect cell, whereby the polypeptide can be expressed. Examples of the gene transfer vector used in the above method include pVL1392, pVL1393, and pBlueBacfI (all manufactured by Invitorogen Corporation).
As a method for co-introducing a recombinant gene transfer vector and a baculovirus into an insect cell for constructing the recombinant virus, for example, a calcium phosphate method (Japanese Unexamined Patent Publication No. H2-227075), a lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), or the like can be mentioned.
The recombinant vector according to the present invention preferably further contains a selection marker gene for selecting a transformant. For example, in Escherichia coli, resistance genes for various drugs such as tetracycline, ampicillin, and kanamycin can be used as selection marker genes. A recessive selection marker capable of complementing a genetic mutation involved in auxotrophy can also be used. In yeast, a resistance gene for geneticin can be used as a selection marker gene, and a gene complementing a genetic mutation involved in auxotrophy, or a selection marker such as LEU2, URA3, TRPI, or HIS3 can also be used. Examples of the selection marker gene for filamentous fungi include a marker gene selected from the group consisting of 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)), and a leucine auxotrophy-complementing gene. Further, in the case where the host is an auxotrophic mutant strain, a wild-type gene complementing the auxotrophy can also be used as a selection marker gene.
The selection of the host transformed with the expression vector according to the present invention can be carried out by plaque hybridization and colony hybridization using a probe that selectively binds to the nucleic acid according to the present invention. As the probe, it is possible to use a probe obtained by modifying a partial DNA fragment amplified by a PCR method based on sequence information of the nucleic acid according to the present invention with a radioisotope or digoxigenin.
(Production of Modified Fibroin)
In the host transformed with the expression vector according to the present invention, the modified fibroin according to the present invention can be produced by expressing the nucleic acid according to the present invention. As for the expression method, secretory production, fusion protein expression, or the like, in addition to direct expression, can be carried out according to the method described in Molecular Cloning, 2nd edition. In the case where it is expressed by yeast, an animal cell, or an insect cell, a modified fibroin can be obtained as a polypeptide to which a sugar or sugar chain is added.
The modified fibroin according to the present invention can be produced, for example, by culturing a host transformed with the expression vector according to the present invention in a culture medium, producing and accumulating the modified fibroin according to the present invention in the culture medium, and then collecting the modified fibroin from the culture medium. The method for culturing the host according to the present invention in a culture medium can be carried out according to a method commonly used for culturing a host.
In the case where the host according to the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, any of a natural medium and a synthetic medium may be used as a culture medium of the host according to the present invention as long as it contains a carbon source, a nitrogen source, inorganic salts and the like which can be assimilated by the host and it is capable of efficiently culturing the host.
As the carbon source, any carbon source that can be assimilated by the host may be used. Examples of the carbon source that can be used include carbohydrates such as glucose, fructose, sucrose, and molasses, starch and starch hydrolyzates containing them, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
Examples of the nitrogen source that can be 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, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean cake and soybean cake hydrolyzate, various fermented microbial cells and digested products thereof.
Examples of the inorganic salt that can be used include potassium dihydrogen phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
Culture of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15° C. to 40° C. The culture time is usually 16 hours to 7 days. It is preferable to maintain the pH of the culture medium during the culture at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like.
In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary during the culture. In the case of culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, in the case of culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-3-D-thiogalactopyranoside or the like is used, and in the case of culturing a microorganism transformed with an expression vector using a trp promoter, indole acrylic acid or the like may be added to the medium.
As a culture medium for insect cells, commonly used TNM-FH medium (manufactured by Pharmingen Inc.), Sf-900 II SFM medium (manufactured by Life Technologies Corporation), ExCell 400 and ExCell 405 (both manufactured by JRH Biosciences Inc.), Grace's Insect Medium (Nature, 195, 788 (1962)), and the like can be used.
Culture of insect cells can be carried out, for example, for a culture time of 1 to 5 days under conditions such as pH 6 to 7 of culture medium and culture temperature 25° C. to 30° C. In addition, an antibiotic such as gentamicin may be added to the culture medium as necessary during the culture.
In the case where the host is a plant cell, the transformed plant cell may be directly cultured, or it may be differentiated into a plant organ and then cultured. As the culture medium for culturing a plant cell, for example, commonly used Murashige and Skoog (MS) medium, White medium, or a medium in which a plant hormone such as auxin or cytokinin is added to these media can be used.
Culture of animal cells can be carried out, for example, for a culture time of 3 to 60 days under conditions such as pH 5 to 9 of the culture medium and culture temperature 20° C. to 40° C. In addition, an antibiotic such as kanamycin or hygromycin may be added to the medium as necessary during the culture.
As a method for producing a modified fibroin using a host transformed with the expression vector according to the present invention, there are a method for producing the modified fibroin in 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 to be used and the structure of the modified fibroin to be produced.
For example, in the case where a modified fibroin is produced in the host cell or on the outer membrane of the host cell, the production method can be altered to actively secrete the modified fibroin outside the host cell according to the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Lowe et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)), or the methods described in Japanese Unexamined Patent Publication No. H5-336963, International Publication No. WO94/23021, and the like. That is, the modified fibroin can be actively secreted outside the host cell by expressing the modified fibroin in a form in which a signal peptide is added to a polypeptide containing an active site of a modified fibroin using a gene recombination technique.
The modified fibroin produced by the host transformed with the expression vector according to the present invention can be isolated and purified by a method commonly used for protein isolation and purification. For example, in the case where the modified fibroin is expressed in a dissolved state in cells, the host cells are recovered by centrifugation after completion of the culture, suspended in an aqueous buffer solution, and then disrupted using an ultrasonicator, a French press, a Manton-Gaulin homogenizer, a Dyno-Mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified preparation can be obtained by a method commonly used for protein isolation and purification, that is, a solvent extraction method, a salting-out method using ammonium sulfate or the like, a desalting method, a precipitation method using an organic solvent, an anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Kasei Kogyo Kabushiki Kaisha), an cation exchange chromatography method using a resin such as S-Sepharose FF (Pharmacia Corporation), a hydrophobic chromatography method using a resin such as butyl sepharose or phenyl sepharose, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing or the like, alone or in combination thereof.
As the chromatography, column chromatography using phenyl-TOYOPEARL (available from Tosoh Corporation), DEAE-TOYOPEARL (available from Tosoh Corporation), and Sephadex G-150 (available from Pharmacia Biotech Inc.) is preferably used.
In the case where the modified fibroin is expressed by the formation of an insoluble matter in the cell, similarly, the host cells are recovered, disrupted and centrifuged to recover the insoluble matter of the modified fibroin as a precipitated fraction. The recovered insoluble matter of the modified fibroin can be solubilized with a protein denaturing agent. After this operation, a purified preparation of modified, fibroin can be obtained by the same isolation and purification method as described above.
In the case where a modified fibroin or a derivative in which a sugar chain has been added to the modified fibroin is secreted extracellularly, the modified fibroin or the derivative thereof can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
[Manufacturing Method of Molded Article]
The molded article according to the present embodiment may be manufactured from a molding composition containing the modified fibroin according to the present embodiment using a press molding apparatus.
The heating is preferably carried out at 80° C. to 300° C., more preferably at 100° C. to 180° C., and still more preferably at 100° C. to 130° C. The pressurization is preferably carried out at 5 kN or more, more preferably at 10 kN or more, and still more preferably at 20 kN or more. Further, the time during which the treatment is continued after a predetermined heating and pressurizing condition has been fulfilled (temperature retention condition) is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, and still more preferably 5 to 30 minutes.
The molding composition containing the modified fibroin may contain only modified fibroin or may further contain optional additives. By including an additive, a molding composition having various different physical properties can be produced. Examples of the additives include softeners, crystal nucleating agents, foaming agents, weathering agents, hydrolysis resistors, reducing agents, colorants, modifiers, stabilizers, lubricants, fillers, flame retardants, reinforcing agents, mold release agents, antistatic agents, and antioxidants.
Examples of the softening agent include high molecular weight alcohols such as glycerin and polyethylene glycol, and sugars such as starch, water, surfactants.
Examples of the crystal nucleating agents include kaolin, talc, montmorilloid, magnesium oxide, aluminum oxide, and other types of polyamides.
Examples of the foaming agent include sodium bicarbonate plus acid or alkali, acid-added aluminum powder, water-added isocyanate with, and the like.
Examples of the weathering agent include benzotriazole, benzophenone, triazine and the like can be mentioned.
Examples of the hydrolysis resistant agent include epoxy reagent, carbodiimide, isocyanate, and oxazoline.
[Product]
The fibroin fiber formed from the modified fibroin according to the present invention can be applied as a fiber or a yarn to a woven fabric, a knitted fabric, a braided fabric, and a nonwoven fabric. Such a fibroin fiber can also be applied to high strength applications such as ropes, surgical sutures, flexible stops for electrical components, and physiologically active materials for implantation (for example, artificial ligament and aortic band).
The modified fibroin according to the present invention can also be applied to filaments, films, foams, spheres, nanofibrils, hydrogels, resins and equivalents thereof; which can be produced in accordance with the method described in Japanese Unexamined Patent Publication No. 2009-505668, Japanese Unexamined Patent Publication No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735, or the like.
Hereinafter, the present invention will be described more specifically with respect 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 nucleotide sequence and amino acid sequence of Nephila clavipes (GenBank Accession Number: P46804.1, GI: 1174415) which is naturally occurring fibroin, fibroins and modified fibroins having amino acid sequences set forth in SEQ ID NOs: 1 to 5 and 7 to 11 were designed.
The amino acid sequence set forth in SEQ ID NO: 1 is a sequence obtained by deleting alanine residues of an amino acid sequence in which the alanine residues in the (A)n motif of the naturally occurring fibroin are consecutive so that the number of consecutive alanine residues is 5. The amino acid sequence set forth in SEQ ID NO: 7 is a sequence obtained by adding the amino acid sequence set forth in SEQ ID NO: 6 (tag sequence and hinge sequence) to the N-terminal of the amino acid sequence set forth in SEQ ID NO: 1 (Comparative Example 1).
The amino acid sequence set forth in SEQ ID NO: 2 is a sequence obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 1, and deleting a part of the amino acid on the N-terminal side such that the molecular weight thereof is set to be approximately the same as that of the amino acid sequence set forth in SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 8 is a sequence obtained by adding the amino acid sequence set forth in SEQ ID NO: 6 (tag sequence and hinge sequence) to the N-terminal of the amino acid sequence set forth in SEQ ID NO: 2 (Example 1).
The amino acid sequence set forth in SEQ ID NO: 3 is a sequence obtained by inserting two alanine residues at the C-terminal side of each (A)n motif of the amino acid sequence set forth in SEQ ID NO: 1 and further substituting a part of glutamine (Q) residues with serine (S) residues and deleting a part of amino acids on the N-terminal side such that the molecular weight thereof is set to be approximately the same as the molecular weight of SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 9 is a sequence obtained by adding the amino acid sequence set forth in SEQ ID NO: 6 (tag sequence and hinge sequence) to the N-terminal of the amino acid sequence set forth in SEQ ID NO: 2. (Comparative Example 2)
The amino acid sequence set forth in SEQ ID NO: 4 is a sequence obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at one site for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 3. The amino acid sequence set forth in SEQ ID NO: 10 is a sequence obtained by adding the amino acid sequence set forth in SEQ ID NO: 6 (tag sequence and hinge sequence) to the N-terminal of the amino acid sequence set forth in SEQ ID NO: 4 (Example 2).
The amino acid sequence set forth in SEQ ID NO: 5 is a sequence obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites for each REP with respect to the amino acid sequence set forth in SEQ ID NO: 3. The amino acid sequence set forth in SEQ ID NO: 11 is a sequence obtained by adding the amino acid sequence set forth in SEQ ID NO: 6 (tag sequence and hinge sequence) to the N-terminal of the amino acid sequence set forth in SEQ ID NO: 5 (Example 3).
Each of nucleic acids encoding proteins having amino acid sequences set forth in SEQ ID NOs: 7 to 11 in which a His tag sequence and a hinge sequence (SEQ ID NO: 6) have been added to the N-terminal of each designed amino acid sequence set forth in SEQ ID NOs: 1 to 5 was synthesized. In the nucleic acid, an NdeI site was added to the 5′ end and an EcoRI site was added downstream of the stop codon. These five kinds of nucleic acids were cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoR, and then recombined into a protein expression vector pET-22b(+) to obtain an expression vector.
[(2) Expression of Protein]
Escherichia coli BLR (DE3) was transformed with a pET22b(+) expression vector including each of nucleic acids encoding proteins having the amino acid sequences set forth in SEQ ID NOs: 7 to 11. The transformed Escherichia coli was cultured in 2 mL of an LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium (Table 2) containing ampicillin so that the OD600 was 0.005. The temperature of the culture solution was maintained at 30° C. and the flask culture was carried out (for about 15 hours) until the OD600 reached 5, thereby obtaining a seed culture solution.
The seed culture solution was added to a jar fermenter to which 500 ml of a production medium (Table 3) had been added so that the OD600 was 0.05, and the transformed Escherichia coli was inoculated. The culture was carried out while controlling the culture solution temperature at 37° C. and keeping the pH constant at 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
Immediately after glucose in the production medium was completely consumed, a feed solution (455 g/1 L of glucose and 120 g/1 L of Yeast Extract) was added at a rate of 1 ml/min. The culture was carried out while controlling the culture solution temperature at 37° C. and keeping the pH constant at 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Thereafter, 1 M isopropyl-p-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the target protein. Twenty hours after addition of IPTG; the culture solution was centrifuged to recover the bacterial cells. SDS-PAGE was carried out using the bacterial cells prepared from the culture solution before the addition of IPTG and after the addition of IPT, and the expression of the target protein was confirmed by the appearance of a band of a target protein size depending on the addition of IPTG
[(3) Purification of Protein]
The bacterial cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer solution (pH 7.4). The bacterial cells after washing were suspended in 20 mM Tris-HCl buffer solution (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (available from GEA Niro Soavi SpA). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer solution (pH 7.4) until high purity. The precipitate after washing was suspended in 8 M guanidine buffer solution (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg/mL, and dissolved by stirring with a stirrer at 60° C. for 30 minutes. After dissolution, dialysis was carried out with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, the water content was removed with a freeze dryer, and the freeze-dried powder was recovered.
The degree of purification of the target protein in the freeze-dried powder thus obtained was confirmed by image analysis of polyacrylamide gel electrophoresis results of the powder using TotalLab (Nonlinear Dynamics Ltd.). As a result, the purity of each protein was about 85%. The production amount of each target protein calculated from the weight of the freeze-dried powder is shown in Tables 4 and 5 as relative values when the value of GEN495 (SEQ ID NO: 7: Comparative Example 1) is taken as 100.
[(4) Manufacture of Molded Article]
1.35 g of each of the freeze-dried powders (hereinafter referred to as “samples”) obtained in the above [(3) Purification of protein] was weighed out, and this sample was placed in the mold 2 of the press molding apparatus 10 shown in
[(5) Measurement of Bending Strength]
The obtained molded article was allowed to stand for 1 day under a condition of 20° C. and 65% in a thermo-hygrostat (Leck LHL-113, manufactured by espec), then using an autograph (AG-Xplus, manufactured by Shimadzu Corporation) and basket jig, a three point bending test was conducted. A load cell with a rated capacity of 50 kN was used. At this time, the distance between fulcrums of three point bending was fixed at 27 mm and the measurement speed was set to 1 mm/min. The size of the molded article was measured with a micro caliper. The results of the bending strength measurement test are shown in Table 4 as relative values when the value of GEN 495 (SEQ ID NO: 7: Comparative Example 1) is taken as 100.
The modified fibroins having an amino acid sequence containing a region with locally high hydropathy index in REP showed remarkably improved productivity (Examples 1 to 3), and a molded article produced from the modified fibroins showed a remarkably improved bending strength (Example 1).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-124674 | Jun 2016 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/023116 | 6/23/2017 | WO | 00 |
