Patent application title: DRAGLINE PROTEIN
Inventors:
Tianfu Zhao (Kitakatsuragi-Gun, JP)
Yujun Wang (Kitakatsuragi-Gun, JP)
Masao Nakagaki (Ueda-Shi, JP)
IPC8 Class: AC07K14435FI
USPC Class:
530353
Class name: Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof proteins, i.e., more than 100 amino acid residues scleroproteins, e.g., fibroin, elastin, silk, etc.
Publication date: 2013-10-10
Patent application number: 20130267682
Abstract:
To provide a new natural fiber material with excellent physical
properties. Any one of the following nucleic acids (a) to (d): (a) a
nucleic acid having a base sequence of SEQ ID NO: 1 or 19; (b) a nucleic
acid encoding a protein having an amino acid sequence of SEQ ID NO: 2 or
20; (c) a nucleic acid encoding a dragline protein and having a sequence
identity of 90% or more with the nucleic acid (a); (d) a nucleic acid
which encodes a dragline protein and hybridizes with a complementary
chain of the nucleic acid (a) under stringent conditions.Claims:
1. Any one of the following nucleic acids (a) to (d): (a) a nucleic acid
having a base sequence of SEQ ID NO: 1 or 19; (b) a nucleic acid encoding
a protein having an amino acid sequence of SEQ ID NO: 2 or 20; (c) a
nucleic acid encoding a dragline protein and having a sequence identity
of 90% or more with the nucleic acid (a); (d) a nucleic acid which
encodes a dragline protein and hybridizes with a complementary chain of
the nucleic acid (a) under stringent conditions.
2. A nucleic acid encoding a dragline protein, comprising any one of the following nucleic acids (e) to (h) and having a sequence identity of 70% or more with a nucleic acid having the base sequence of SEQ ID NO: 1: (e) a nucleic acid having a base sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17; (f) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18; (g) a nucleic acid having a sequence identity of 90% or more with the nucleic acid (e); (h) a nucleic acid which hybridizes with a complementary chain of the nucleic acid (e) under stringent conditions.
3. The nucleic acid according to claim 2, having a sequence identity of 80% or more with a nucleic acid having the base sequence of SEQ ID NO: 1.
4. A nucleic acid encoding a dragline protein, comprising any one of the following nucleic acids (i) to (l) and having a sequence identity of 70% or more with a nucleic acid having the base sequence of SEQ ID NO: 19: (i) a nucleic acid having a base sequence of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 or 35; (j) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36; (k) a nucleic acid having a sequence identity of 90% or more with the nucleic acid (i); (l) a nucleic acid which hybridizes with a complementary chain of the nucleic acid (i) under stringent conditions.
5. The nucleic acid according to claim 4, having a sequence identity of 80% or more with a nucleic acid having a base sequence of SEQ ID NO: 19.
6. An isolated protein encoded by one of the following nucleic acids (i)-(iv): (i) a nucleic acid having the base sequence of SEQ ID NO: 19; (ii) a nucleic acid encoding a protein having the amino acid sequence of SEQ ID NO: 20; (iii) a nucleic acid encoding a dragline protein with a base sequence identity of 90% or more to SEQ ID NO: 19; or (iv) a nucleic acid which encodes a dragline protein and hybridizes with a complementary chain of the nucleic acid of SEQ ID NO: 19 under stringent conditions including hybridization in a buffer solution of 50% formamide, 1M NaCl, 1% SDS at 37.degree. C. and washing with 0.1.times.SSC at 60.degree. C.
7. A recombinant organism having the nucleic acid according to claim 1 introduced therein.
8. A recombinant silkworm having the nucleic acid according to claim 1 introduced therein.
9. A protein according to claim 6, which is produced by a recombinant organism.
10. An isolated silk thread containing the protein according to claim 6, produced by a recombinant silkworm.
11. An isolated dragline protein having an amino acid sequence selected from the group consisting of: (i) the amino acid sequence of SEQ ID NO: 20; and (ii) an amino acid sequence having a sequence identity of 90% or more with SEQ ID NO: 20.
12. An isolated dragline protein having the following amino acid sequence (i) or (ii) and having a sequence identity of 70% or more with the amino acid sequence of SEQ ID NO: 20, (i) an amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36; (ii) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence (i).
13. The dragline protein according to claim 12, wherein the amino acid sequence has a sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 20.
14. A protein having an amino acid sequence represented by the following formula (1) or an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by the formula (1): [X1-X2-X3-(X4)m-(X5)m-(X6)m-X7-X8]n (1) where each m independently represent an integer of 0 or 1; n represents an integer of 1 to 10; X1 represents any one of amino acid sequences of SEQ ID NO: 37 to 45; X2 represents any one of amino acid sequences of SEQ ID NO: 46 to 52; X3 represents any one of amino acid sequences of SEQ ID NO: 53 to 59; X4 represents an amino acid sequence of SEQ ID NO: 49; X5 represents an amino acid sequence of SEQ ID NO: 60 or 61; X6 represents any one of amino acid sequences of SEQ ID NO: 62 to 64; X7 represents any one of amino acid sequences of SEQ ID NO: 65 to 70; and X8 represents any one of amino acid sequences of SEQ ID NO: 71 to 81.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application Ser. No. 13/226,157 (allowed) filed on Sep. 6, 2011, which claims priority based on Japanese Patent Application No. 2010-203556 filed on Sep. 10, 2010, the contents of which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a nucleic acid, a protein encoded by the nucleic acid, a recombinant organism having the nucleic acid introduced therein and a protein produced by the recombinant organism.
[0004] 2. Related Background Art
[0005] A spider silk is known as a naturally occurring high performance polymer with excellent toughness due to a combination of strength and elasticity. A spider has at most 7 specialized glands, from which many types of spider silk different in nature are produced, and among them, attention has been focused on a dragline produced by Major Ampullate as the toughest spider silk in the development of new materials used in various industrial fields such as medical, aviation and apparel industries.
[0006] A protein called Major Ampullate Spidroin (MaSp) is known as a major protein constituting a dragline, and up to now, gene sequences encoding MaSp proteins of various spiders such as Latrodectus hesperus, Latrodectus geometricus and Nephila clavipes have been elucidated (Non-Patent Literature 1: Nadia A. Ayoub et al., Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes, 2007, Issue 6, e514; Non-Patent Literature 2: William A. Gaines I V et al., Identification and Characterization of Multiple Spidroin 1 Genes Encoding Major Ampullate Silk Proteins in Nephila clavipes, Insect Mol Biol, 2008, 17(5), 465-474; and so on.).
SUMMARY OF THE INVENTION
[0007] However, in each of the industrial fields, demand for a natural fiber with excellent physical properties is increasing more and more and further development of new materials have been expected.
[0008] Then, the present invention aims to provide a material with excellent physical properties for a natural fiber.
[0009] The present inventors intensively studied with a view to achieving the aforementioned aims, as a result, found that a gene encoding an MaSp protein constituting a dragline of Nephila pillipes has a unique structure different from MaSp genes conventionally known, and thereby completed the present invention.
[0010] Thus, the present invention relates to (l) any one of the following nucleic acids (a) to (d):
[0011] (a) a nucleic acid having a base sequence of SEQ ID NO: 1 or 19;
[0012] (b) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 2 or 20;
[0013] (c) a nucleic acid encoding a dragline protein and having a sequence identity of 90% or more with the nucleic acid (a);
[0014] (d) a nucleic acid which encodes a dragline protein and hybridizes with a complementary chain of the nucleic acid (a) under stringent conditions.
[0015] Furthermore, the present invention relates to (2) a nucleic acid encoding a dragline protein, comprising any one of the following nucleic acids (e) to (h) and having a sequence identity of 70% or more, preferably 80% or more, with a nucleic acid having a base sequence of SEQ ID NO: 1:
[0016] (e) a nucleic acid having a base sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17;
[0017] (f) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18;
[0018] (g) a nucleic acid having a sequence identity of 90% or more with the nucleic acid (e);
[0019] (h) a nucleic acid which hybridizes with a complementary chain of the nucleic acid (e) under stringent conditions.
[0020] Furthermore, the present invention relates to (3) the nucleic acid according to (2), having a sequence identity of 80% or more with a nucleic acid having the base sequence of SEQ ID NO: 1.
[0021] Furthermore, the present invention relates to (4) a nucleic acid encoding a dragline protein, comprising any one of the following nucleic acids (i) to (l) and having a sequence identity of 70% or more, preferably 80% or more, with a nucleic acid having a base sequence of SEQ ID NO: 19 and:
[0022] (i) a nucleic acid having a base sequence of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 or 35;
[0023] (j) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36;
[0024] (k) a nucleic acid having a sequence identity of 90% or more with the nucleic acid (i);
[0025] (l) a nucleic acid which hybridizes with a complementary chain of the nucleic acid (i) under stringent conditions.
[0026] Furthermore, the present invention relates to (5) the nucleic acid according to (4), having a sequence identity of 80% or more with a nucleic acid having a base sequence of SEQ ID NO: 19.
[0027] Furthermore, the present invention relates to (6) a protein encoded by the nucleic acid according to any one of (1) to (5).
[0028] By the aforementioned specific nucleic acid according to the present invention, an MaSp protein (dragline protein) with excellent physical properties different from conventional MaSp proteins is coded and the provision of a new material of a natural fiber becomes possible. Particularly, a dragline protein encoded by the nucleic acid of the present invention (the protein according to the present invention) has more excellent elasticity (or resiliency, stretchability, degree of elongation, flexibility) than a conventional one, and preferably employed in various industrial fields, more specifically, in uses requiring elasticity such as medical products and apparel products.
[0029] Furthermore, the present invention relates to (7) a recombinant organism having the nucleic acid according to any one of (1) to (5) introduced therein and (9) a protein produced by the recombinant organism according to (7). According to the recombinant organism of the present invention, a large amount of dragline protein with excellent physical properties encoded by the nucleic acid can be produced. Proteins produced by the recombinant organism can be preferably used in various industrial fields, since they comprise a dragline protein with excellent physical properties.
[0030] In particular, the present invention relates to (8) a recombinant silkworm having the nucleic acid according to any one of (1) to (5) introduced therein and (10) a silk thread produced by the recombinant silkworm according to (8). According to the recombinant silkworm of the present invention, a large amount of silk thread comprising a dragline protein with excellent physical properties encoded by the nucleic acid can be produced. The silk thread produced by the recombinant silkworm has more excellent physical properties than conventional silk threads, and particularly has excellent elasticity, since it comprises a dragline protein with excellent physical properties.
[0031] Furthermore, the present invention relates to (11) a dragline protein having an amino acid sequence (m) or (n):
[0032] (m) an amino acid sequence of SEQ ID NO: 2 or 20;
[0033] (n) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence (m).
[0034] Furthermore, the present invention relates to (12) a dragline protein having the following amino acid sequence (o) or (p):
[0035] (o) an amino acid sequence having the following amino acid sequence (o1) or (o2) and having a sequence identity of 70% or more, preferably 80% or more, with the amino acid sequence of SEQ ID NO: 2;
[0036] (o1) an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18;
[0037] (o2) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence (o1);
[0038] (p) an amino acid sequence having the following amino acid sequence (p1) or (p2) and having a sequence identity of 70% or more, preferably 80% or more, with the amino acid sequence of SEQ ID NO: 20;
[0039] (p1) an amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36;
[0040] (p2) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence (p1).
[0041] Furthermore, the present invention relates to (13) the dragline fiber protein according to (12), wherein the amino acid sequence (o) has a sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence (p) has a sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 20.
[0042] Furthermore, the present invention relates to (14) a protein having an amino acid sequence represented by the following formula (1) or an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by the formula (1):
[X1-X2-X3-(X4)m-(X5)m-(X6)m-X7-X8]n (1).
[0043] In the formula (1), each m independently represent an integer of 0 or 1; n represents an integer of 1 to 10; X1 represents any one of the amino acid sequences of SEQ ID NO: 37 to 45; X2 represents any one of the amino acid sequences of SEQ ID NO: 46 to 52; X3 represents any one of the amino acid sequences of SEQ ID NO: 53 to 59; X4 represents an amino acid sequence of SEQ ID NO: 49; X5 represents an amino acid sequence of SEQ ID NO: 60 or 61; X6 represents any one of the amino acid sequences of SEQ ID NO: 62 to 64; X7 represents any one of the amino acid sequences of SEQ ID NO: 65 to 70; and X8 represents any one of the amino acid sequences of SEQ ID NO: 71 to 81.
[0044] The protein according to the present invention has more excellent physical properties due to its unique structure than conventional dragline proteins and thus preferably used in various industrial fields.
[0045] By a nucleic acid of the present invention, a protein with excellent physical properties is provided. Furthermore, by a recombinant organism of the present invention, a protein with excellent physical properties can be produced in a large amount. Particularly, by a recombinant silkworm of the present invention, a silk thread with excellent physical properties can be produced in a large amount. The dragline protein or silk thread provided by the present invention has particularly excellent elasticity. As described above, according to the present invention, it is possible to provide a new material of a natural fiber with excellent physical properties such as elasticity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a view showing cDNA sequence of NP-dragline protein A (SEQ ID NO: 1).
[0047] FIG. 2 is a view showing an amino acid sequence of NP-dragline protein A (SEQ ID NO: 2).
[0048] FIG. 3 is a photograph showing a result of Northern hybridization.
[0049] FIG. 4 is a view showing cDNA sequence of NP-dragline protein B (SEQ ID NO: 19).
[0050] FIG. 5 is a view showing an amino acid sequence of NP-dragline protein B (SEQ ID NO: 20).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] An embodiment for performing the invention will be described below, if necessary, referring to the accompanying drawings. However, the present invention is not limited to the following embodiment.
[0052] The present invention relates to any one of the following nucleic acids:
[0053] (a) a nucleic acid having a base sequence of SEQ ID NO: 1 or 19;
[0054] (b) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 2 or 20;
[0055] (c) a nucleic acid encoding a dragline protein and having a sequence identity of 90% or more with the nucleic acid (a);
[0056] (d) a nucleic acid which encodes a dragline protein and hybridizes with a complementary chain of the nucleic acid (a) under stringent conditions.
[0057] First, the present invention relates to a nucleic acid (a) having a base sequence of SEQ ID NO: 1 or 19. Both base sequences of SEQ ID NO: 1 and 19 are genes encoding a protein (polypeptide) called Major Ampullate Spidroin (MaSp), which is a main component constituting a dragline of Nephila pilipes of the genus Nephila. In this specification, a protein encoded by a nucleic acid having the base sequence of SEQ ID NO: 1 is called "NP-dragline protein A"; a protein encoded by a nucleic acid having the base sequence of SEQ ID NO: 19 is called "NP-dragline protein B". These nucleic acids (a) are not necessarily those obtained from Nephila pilipes and may be artificially synthesized or obtained from a genomic library or a cDNA library or may be obtained by amplifying each of these nucleic acids by PCR and obtained by digestion with restriction enzymes, as long as a nucleic acid has an base sequence of SEQ ID NO: 1 or 19.
[0058] The nucleic acid of the present invention may be the nucleic acid (b) encoding a protein having an amino acid sequence of SEQ ID NO: 2 or 20. Both amino acid sequences of SEQ ID NO: 2 and 20 are an amino acid sequences that an MaSp protein of Nephila pilipes has. Specifically the amino acid sequence of SEQ ID NO: 2 is an amino acid sequence that NP-dragline protein A has, and the amino acid sequence of SEQ ID NO: 20 is an amino acid sequence that NP-dragline protein B has.
[0059] Furthermore, the nucleic acid of the present invention may be the nucleic acid (c) having a sequence identity of 90% or more with a nucleic acid having a base sequence of SEQ ID NO: 1 or 19, as long as the nucleic acid encodes a dragline protein (MaSp). The sequence identity may be 90% or more, but is preferably 93% or more, more preferably 95% or more and further preferably 98% or more.
[0060] Furthermore, the nucleic acid of the present invention may be the nucleic acid (d) which hybridizes with a complementary chain of a nucleic acid having the base sequence of SEQ ID NO: 1 or 19 under stringent conditions as long as the nucleic acid encodes a dragline protein. Herein, "complementary chain" of a nucleic acid refers to a nucleotide sequence which pairs through hydrogen bonding between nucleic acid bases (for example, T to A, C to G). Furthermore, "hybridize" means to form complementary bonding between complementary chains or form interaction between bases of single-strand nucleic acid molecules.
[0061] Herein, "stringent conditions" refers to conditions under which a complementary chain of a nucleotide chain having a homology with a target sequence preferentially hybridizes with the target sequence and a complementary chain of a nucleotide chain having no homology does not substantially hybridize. The stringent conditions are dependent upon the sequence and vary depending upon various situations. As a sequence becomes longer, specific hybridization thereof occurs at a further higher temperature. Generally, for stringent conditions, a temperature is selected so that it is about 5° C. lower than the thermal melting temperature (Tm) of a specific sequence at a predetermined ion strength and pH. Tm is the temperature at which 50% of complementary nucleotides to a target sequence hybridize with the target sequence in an equilibrium state at a predetermined ion strength, pH and nucleic acid concentration. "Stringent conditions" are dependent upon the sequence and vary depending upon various environmental parameters. A general principle of nucleic acid hybridization can be found in Tijssen (Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, New York).
[0062] Typically, the stringent conditions are those in which the salt concentration is less than about 1.0 M Na.sup.+, typically about 0.01 to 1.0 M of Na.sup.+ concentration (or another salt) at pH 7.0 to 8.3; and the temperature is at least about 30° C. for a short nucleotide (for example, 10 to 50 nucleotides) and at least about 60° C. for a long nucleotide (for example, longer than 50 nucleotides). The stringent conditions can be also achieved by addition of an unstablizing agent such as formamide. The stringent conditions referred in this specification include hybridization in a buffer solution of 50% formamide, 1M NaCl, 1% SDS (37° C.) and washing with 0.1×SSC at 60° C.
[0063] The nucleic acid of the present invention may be a nucleic acid having a sequence identity of 70% or more with a nucleic acid having the base sequence of SEQ ID NO: 1 as long as it comprises any one of the following nucleic acids (e) to (h) and encodes a dragline protein. The sequence identity is satisfactorily 70% or more, preferably 75% or more, more preferably 80% or more, further preferably 85% or more, and particularly preferably 88% or more. The nucleic acids are:
[0064] (e) a nucleic acid having a base sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17;
[0065] (f) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18;
[0066] (g) a nucleic acid having a sequence identity of 90% or more with the nucleic acid (e);
[0067] (h) a nucleic acid which hybridizes with a complementary chain of the nucleic acid (e) under stringent conditions.
[0068] In the base sequence of SEQ ID NO: 1, base sequences of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 and 17 are a sequences which have an important characteristics for encoding a dragline protein of the present invention with excellent physical properties. By the inclusion of the nucleic acid having such a characteristic sequence, even a nucleic acid having a sequence identity of only 70% or more with a nucleic acid having the base sequence of SEQ ID NO: 1 is made capable of encoding a dragline protein of the present invention with excellent physical properties as well as the nucleic acid having the base sequence of SEQ ID NO: 1.
[0069] A protein having an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 is a protein encoded by a base sequences of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17, respectively.
[0070] The sequence identity of the nucleic acid (g) with the nucleic acid (e) may be 90% or more, but is preferably 93% or more, more preferably 95% or more and further preferably 98% or more.
[0071] Furthermore, the nucleic acid of the present invention may be a nucleic acid having a sequence identity of 70% or more with a nucleic acid having the base sequence of SEQ ID NO: 19 as long as it comprises any one of the following nucleic acids (i) to (l) and encodes a dragline protein. The sequence identity may be 70% or more, but is preferably 75% or more, more preferably 80% or more, further preferably 85% or more and particularly preferably 88% or more. The nucleic acids are:
[0072] (i) a nucleic acid having a base sequence of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 or 35;
[0073] (j) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36;
[0074] (k) a nucleic acid having a sequence identity of 90% or more with the nucleic acid (i);
[0075] (l) a nucleic acid which hybridizes with a complementary chain of the nucleic acid (i) under stringent conditions.
[0076] In the base sequence of SEQ ID NO: 19, base sequences of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 or 35 are sequences which have an important characteristics for encoding a dragline protein of the present invention with excellent physical properties. By the inclusion of the nucleic acid having such a characteristic sequence, even a nucleic acid having a sequence identity of only 70% or more with the nucleic acid having the base sequence of SEQ ID NO: 19 can be made capable of encoding a dragline protein with excellent physical properties of the present invention as well as the nucleic acid having the base sequence of SEQ ID NO: 19.
[0077] A protein having an amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36 is a protein encoded by a base sequences of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 or 35, respectively.
[0078] The sequence identity of the nucleic acid (k) with the nucleic acid (i) may be 90% or more, but is preferably 93% or more, more preferably 95% or more and further preferably 98% or more.
[0079] Furthermore, the present invention relates to a recombinant organism into which a nucleic acid of the present invention as mentioned above is introduced and a protein produced by the recombinant organism. Particularly, the present invention relates to a recombinant silkworm into which a nucleic acid of the present invention as mentioned above is introduced and a silk thread produced by the recombinant silkworm.
[0080] In this specification, "recombinant organism" refers to an organism transformed by introducing a foreign gene into the chromosome by means of genetic recombination. The organism to be transformed is not particularly limited and, for example, an insect, an animal, a plant or a microorganism may be used; however, an insect is preferred. Examples of the preferable insect include Bombyx mori, Bombyx mandarina, Antheraea yamamai and Antheraea pernyi. Among them, Bombyx mori and Bombyx mandarina belonging to Bombycidae are preferably used, and Bombyx mori is particularly preferably used.
[0081] In this specification, "silkworm" refers to Bombyx mori. A silkworm may be either a breed for experimentation or a commercial breed commercialized for practical use. Furthermore, "recombinant silkworm" refers to a silkworm transformed by introducing a foreign gene into the silkworm chromosome by means of genetic recombination. Genetic recombination is performed by a method, for example, using a transposon; however, the method is not limited and any method is used as long as it can introduce a foreign gene into a silkworm and recombination of a gene can be performed by other methods including electroporation.
[0082] In this specification, "silk thread" is a fiber, which is ejected by Bombyx mori, constituting a cocoon and comprising a fibroin protein as a main component. The fibroin protein is composed of two large and small subunits (H-chain and L chain).
[0083] In this specification, "Nephila pilipes" refers to Nephila pilipes of the genus Nephila without particularly limiting their growing district.
[0084] Furthermore, the present invention relates to a dragline protein having the following amino acid sequences (m) or (n):
[0085] (m) an amino acid sequence of SEQ ID NO: 2 or 20;
[0086] (n) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence (m).
[0087] The sequence identity of the amino acid sequence (n) with the amino acid sequence (m) may be 90% or more, but is preferably 93% or more, more preferably 95% or more and further preferably 98% or more.
[0088] Furthermore, the present invention relates to a dragline protein having the following amino acid sequence (o) or (p):
[0089] (o) an amino acid sequence having the following amino acid sequence (o1) or (o2) and having a sequence identity of 70% or more with the amino acid sequence of SEQ ID NO: 2;
[0090] (o1) an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18;
[0091] (o2) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence (o1);
[0092] (p) an amino acid sequence having the following amino acid sequence (p1) or (p2) and having a sequence identity of 70% or more with the amino acid sequence of SEQ ID NO: 20;
[0093] (p1) an amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36;
[0094] (p2) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence (p1).
[0095] The sequence identity of the amino acid sequence (o) with the amino acid sequence of SEQ ID NO: 2 may be 70% or more, but is preferably 75% or more, more preferably 80% or more, further preferably 85% or more and particularly preferably 88% or more.
[0096] Similarly, the sequence identity of the amino acid sequence (p) with the amino acid sequence of SEQ ID NO: 20 is satisfactorily 70% or more, preferably 75% or more, more preferably 80% or more, further preferably 85% or more and particularly preferably 88% or more.
[0097] Furthermore, the sequence identity of the amino acid sequence (o2) with the amino acid sequence (o1) is satisfactorily 90% or more, preferably 93% or more, more preferably 95% or more and further preferably 98% or more.
[0098] Similarly, the sequence identity of the amino acid sequence (p2) with the amino acid sequence (p1) is satisfactorily 90% or more, preferably 93% or more, more preferably 95% or more and further preferably 98% or more.
[0099] Furthermore, the present invention relates to a protein having an amino acid sequence represented by the following formula (1):
[X1-X2-X3-(X4)m-(X5)m-(X6)m-X7-X8]n (1)
[0100] An amino acid sequence represented by the formula (1) has the "n" number of repeat units represented by [X1-X2-X3-(X4)m-(X5)m-(X6)m-X7-X8]. The number "n" of repeat units is not particularly limited; however, the number is preferably 1 to 10, more preferably 2 to 9, further preferably 3 to 8, and particularly preferably n=8.
[0101] In the formula (1), each m independently represent an integer of 0 or 1. More specifically, there is a repeat unit having an amino acid sequence represented by X4, X5 or X6 and a repeat unit having no such an amino acid sequence.
[0102] In the formula (1), X1 represents any one of the amino acid sequences of SEQ ID NO: 37 to 45; X2 represents any one of the amino acid sequences of SEQ ID NO: 46 to 52; X3 represents any one of the amino acid sequences of SEQ ID NO: 53 to 59; X4 represents an amino acid sequence of SEQ ID NO: 49; X5 represents an amino acid sequence of SEQ ID NO: 60 or 61; X6 represents any one of the amino acid sequences of SEQ ID NO: 62 to 64; X7 represents any one of the amino acid sequences of SEQ ID NO: 65 to 70; and X8 represents any one of the amino acid sequences of SEQ ID NO: 71 to 81.
[0103] Furthermore, the protein according to the present invention may be a protein having an amino acid sequence having a sequence identity of 90% or more with an amino acid sequence represented by the formula (1). The sequence identity may be 90% or more, but is preferably 93% or more, more preferably 95% or more and further preferably 98% or more.
[0104] FIG. 1 is a view showing cDNA sequence of NP-dragline protein A, which is an MaSp protein of Nephila pilipes. The gene sequence shown in FIG. 1 is identical with the base sequence of SEQ ID NO: 1.
[0105] FIG. 2 is a view showing the amino acid sequence of NP-dragline protein A encoded by a nucleic acid having the gene sequence (base sequence of SEQ ID NO: 1) shown in FIG. 1. The amino acid sequence shown in FIG. 2 is identical with the amino acid sequence of SEQ ID NO: 2.
[0106] Furthermore, FIG. 4 is a view showing cDNA sequence of NP-dragline protein B, which is another MaSp protein of Nephila pilipes. The gene sequence shown in FIG. 4 is identical with the base sequence of SEQ ID NO: 19.
[0107] FIG. 5 is a view showing the amino acid sequence of NP-dragline protein B encoded by a nucleic acid having the gene sequence (base sequence of SEQ ID NO: 19) shown in FIG. 4. The amino acid sequence shown in FIG. 5 is identical with the amino acid sequence of SEQ ID NO: 20.
[0108] As shown in FIG. 2 or FIG. 5, the dragline protein encoded by a nucleic acid having a base sequence of SEQ ID NO: 1 or 19 is composed of the amino acid sequence represented by the following the formula (2):
[(α)(V)(β)]q (2)
[0109] The amino acid sequence represented by the formula (2) has the "q" number of repeat units represented by [(α)(V)(β)]. The number "q" of repeat units is not particularly limited; however, the number is satisfactorily 1 to 100, preferably 1 to 10, more preferably 2 to 9, and further preferably 3 to 8, and particularly preferably q=8.
[0110] In the formula (2), (α) is composed of a glycine-rich sequence having 2 to 4 GGX units arranged next to each other and represents an amorphous region where an non-crystalline α-helix structure is formed. (V) represents a paracrystalline region rich in GX content and (β) represents a crystal region rich in alanine or threonine where a β-pleat sheet is formed.
[0111] X comprised in the (α) and (V) is more likely to represent glutamine, alanine, serine, leucine, proline, tyrosine etc., but is not limited to these and may represent a different amino acid other than those mentioned above. Furthermore, it is not necessary that a plurality of X are the same amino acids.
[0112] A specific molecular structure of the dragline protein shown in FIG. 2 or FIG. 5 and the physical properties of a dragline protein obtained by the structure will be described below.
[0113] First, in the (α) region (non-crystalline amorphous region) of the dragline protein shown in FIG. 2 or FIG. 5, 4 units of GGX are arranged next to each other. By virtue of such a sequence, a dragline forms α-helix structure. Usually, α-helix structure is bent in a fiber, but it changes into linear conformation along the fiber axis by stretching. Likewise, upon external stress α-helix structure is drastically elongated and thereby the fiber becomes elastic. On the other hand, in a conventionally known spider dragline protein (MaSp), 4 units of GGX arranged next to each other cannot be found (see Non-Patent Literature 1, 2 etc.). From the above, since a unique structure having 4 units of GGX arranged next to each other is formed in the (α) region, the elasticity (or resiliency, stretchability, degree of elongation, flexibility) of the dragline protein obtained by the present invention conceivably improves.
[0114] A finding that a thread becomes elastic by the presence of a GGX repeat motif is descried in the following documents:
[0115] Cheryl Y. Hayashi et al., Evidence from Flagelliform Silk cDNA for the Structural Basis of Elasticity and Modular Nature of Spider Silks, 1998, p. 779;
[0116] Thomas Scheibel, Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins, 2004, p. 2.
[0117] Furthermore, the (V) region (paracrystalline region) of the dragline protein shown in FIG. 2 or FIG. 5 is rich in a hydrophilic amino acid. As shown in Table 1, the dragline protein shown in FIG. 2 or FIG. 5 is rich in a hydrophilic amino acid compared to a dragline protein of conventionally known Nephila clavipes (North America) and Japanese Nephila clavata. By virtue of this, it is considered that a dragline protein obtained by the present invention increases in hygroscopicity. Furthermore, low crystallinity of a dragline protein is conceivably a factor of increasing hygroscopicity.
[0118] Furthermore, in the (β) region (crystalline region) of the dragline protein shown in FIG. 2 or FIG. 5, polar amino acids such as threonine and asparagine are comprised between polyalanines. Since the dragline protein obtained by the present invention has a polyalanine (Poly(A)) motif rich in polar amino acid, excellent toughness is conceivably obtained.
[0119] The finding that a thread becomes tough by the presence of a polyalanine (Poly(A)) motif rich in polar amino acid is described in the following documents:
[0120] Glareh Askarieh et al., Self-assembly of spider silk proteins is controlled by a pH-sensitive relay, 2010, vol. 465, p. 1;
[0121] J. M. GOSLINE, et al., THE MECHANICAL DESIGN OF SPIDER SILKS: FROM FIBROIN SEQUENCE TO MECHANICAL FUNCTION, 1999, p. 3299.
[0122] Furthermore, as shown in Table 1, the dragline protein shown in FIG. 2 or FIG. 5 comprises a polar amino acid twice as large as in conventionally known Nephila clavipes (North America) and Japanese Nephila clavata. By the presence of the polar amino acid residue present in a large amount within a molecule as mentioned, when external stress is applied, molecules are regularly arranged along the direction of the applied stress to increase interaction force between the molecules. In this manner, the dragline acquires excellent strength. In particular, hydrogen bonding between molecules conceivably plays a role in increasing the strength of a thread fiber.
[0123] Table 1 shows the contents of a polar amino acid and a hydrophilic amino acid in MaSp protein of Nephila pilipes, Nephila clavipes (North America), Japanese Nephila clavata. The content of a polar amino acid represents the content of N (Asn), C (Cys), Q (Gln), S (Ser), T (Thr) and Y (Tyr) and the content of a hydrophilic amino acid represents the contents of R (Arg), N (Asn), D (Asp), Q (Gln), E (Glu), H (His), K (Lys), S (Ser) and T (Thr).
TABLE-US-00001 TABLE 1 Polar amino Hydrophilic amino acid (%) acid (%) Nephila pilipes 31.05 29.41 Nephila clavipes 15.71 14.85 (North America) Japanese Nephila clavata 15.15 11.01
EXAMPLES
[0124] The present invention will be more specifically described by way of Examples. However, the present invention is not limited to the following Examples.
[0125] As a test animal, a female adult spider of Nephila pilipes collected in July was used.
[0126] (RNA Extraction)
[0127] Total RNA was prepared from the Major Ampullate of the spider of Nephila pilipes. The Major Ampullate of the spider was dissected in physiological saline solution (NaCl 0.75%) and TRIZOL (1 ml) was added thereto and sufficiently ground. The resultant suspension solution was separated with chloroform (200 μl) and removed. The water layer was transferred to another tube and the same amount of isopropanol was added thereto to precipitate RNA. The precipitate was rinsed with 75% ethanol and stored at -80° C. Thereafter, it was centrifuged at 7500 rpm, 4° C. for 5 minutes, dried for 8 minutes in vacuum, and dissolved in RNase-free water at 55° C. for 10 minutes and used as a sample. The sample was subjected to agarose electrophoresis to confirm extraction of RNA.
[0128] (Construction of cDNA Library)
[0129] Synthesis and construction of cDNA library of the Major Ampullate by the G-capping method was outsourced to Takara Bio Inc. A library vector (pDNR-LIB) was dissolved in TE (about 50 μl).
[0130] (Cloning and Sequence)
[0131] An electroporation method was employed to perform transformation at a high probability. The cDNA library solution prepared was used as a DNA solution. As competent cells, "Electro MAX® DH12S® Cells" (Cat. No. 18312-017) manufactured by Invitrogen were used. As a cuvette, a cuvette of 0.1 cm in size was used.
[0132] First, a cuvette was cooled on ice in advance. After 50 μl of competent cells (>1010 cfu/μg) in a tube were thawed on ice and 1 μl of the cDNA library solution was added to the tube. The resultant mixture solution was transferred to the cuvette so as to obtain a homogenous state. Conditions of electroporation include a voltage of 2.5 kV, a pulse controller (R2-7) 200Ω, a capacitance of 25 μG. Pulse was applied once and 1 ml of SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgS04, 20 mM glucose) was added to the cuvette as soon as possible to suspend the solution. The suspension solution was transferred to a culture tube and cultured for 1 to 1.5 hours, and thereafter scattered on an LB plate (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl) comprising an antibiotic substance (ampicillin), IPTG and X-Gal. White colony grown in the plate was taken and inoculated on LB (+ampicillin) medium, 588 recombinant plasmids were selected at random and purified by use of FlexiPrep® Kit (manufactured by Amersham plc).
[0133] (Sequence and Comparative Analysis of Sequence)
[0134] The sequence of an insert was analyzed by "ABI Prism genetic analyzer 3100" (manufactured by Life Technologies Corporation) and T7 primer. Computer analysis of DNA and an amino acid sequence was performed by use of "Genetyx package" (manufactured by Genetyx Corporation) and "Sequencher 4.14" (Demo version) (manufactured by Gene Codes Corporation). Sequence comparison was made based on homology analysis of protein data base by means of SIB BLAST Network Service of an ExPASy Proteomics server (http://www.expasy.org).
[0135] (Experiment for Proving Specific Expression of Silk Gland)
[0136] MaSp (major ampullate spidroin) is expressed in the major ampullate as the name implies. To prove that the gene of the present invention works in the major ampullate, a Northern hybridization experiment was performed between a probe, which was prepared by using the 3' end sequence of a cDNA sequence (the C terminal of the amino acid sequence) and RNA samples extracted from 4 silk glands of a spider (flagelliform gland, tubular gland, major ampullate, minor ampullate). FIG. 3 shows the results of the Northern hybridization. To lanes 1 to 4 of FIG. 3, RNA samples extracted from flagelliform gland, tubular gland, major ampullate and minor ampullate were supplied and flow respectively in this order. From the results, it was found that the gene (nucleic acid) of the present invention is specifically expressed in the major ampullate of Nephila pilipes. Furthermore, the molecular weight of the transcribed substance was estimated to be about 3 to 4 kb.
[0137] (Evaluation on Physical Properties of Dragline)
[0138] To compare the dragline of Nephila pilipes and the dragline of a conventionally known spider in physical properties, the degree of elongation (elastic modulus) of each of the fibers was measured. At the day before measurement, sample draglines were allowed to stand still at 20° C., RH65% for 24 hours to adjust the moisture contents thereof. Then, the sample fibers of 20 mm were subjected to an elongation test performed under the conditions: 20° C., RH65% at a stretch rate of 20 mm/min by using a tension tester, "Tensilon UTM-III-100" (manufactured by Toyo Baldwin). As a conventionally known spider, Japanese Nephila clavata and Argiope bruennichi were used. The results are shown in Table 2.
TABLE-US-00002 TABLE 2 Elongation (%) Argiope bruennichi 26.1 Japanese Nephila clavata 22.3 Nephila pilipes 29.4
[0139] As shown in Table 2, it was found that the dragline of Nephila pilipes has excellent elasticity compared to those of conventionally known spiders. More specifically, it was demonstrated that the nucleic acid of the present invention encodes a dragline protein with excellent elasticity.
[0140] The dragline protein provided by the present invention, since it is natural fiber excellent in elasticity, can be preferably used as a new material in various industrial fields such as medical, aviation and apparel industries.
Sequence CWU
1
1
8111000DNANephila pilipes 1gggggatatg gagcaggaag tggatctacc atcgcaataa
ctgctggtgg tcttggtgga 60tctggaggtc aaggtggcca aataccatct ggcgctgttg
gacaaggaac tcaaggatat 120ggaacaggaa gtggagcaac catcgcatta actgctggtg
gacttggggg acaaggtggt 180caaggaccat ctggctctgg tggacaaggc ccatcaggac
aaggagctca aggacctggc 240ggatatggag caggaaatgc agccgccgcc aacgcagcag
ctagtggact tggaggctat 300ggagttggtg ggcagggaag tggccaaaga ccatctggag
ctggtggaca aggtgctcaa 360gcgccaggtg gatatggaac aggaagtgga tcgaccatcg
taataactgc tggtggacag 420agaggacaag gtggtcaagg accatcagga caattagctc
aagcacctag tggatatgga 480caaggaagtg gagccgccgc cgcctctggt ggtcttggag
gatatggagg tcaaggtggc 540caaagatcat ctggcgctgg tgcacaagga actcaaggat
atggtacagg aagtggaaca 600actatcgcat taactgctgg tggtattgga ggatctggag
gtcaaggtgg ccaaagacca 660tctggcattg gtggacaagg agctcaaggg ccaggtggat
atggagcagg aagtggatct 720accatcgcaa taactactgg tggtcttggt ggatatggag
gtcaaggtgg ccaaagacca 780tctggcgctg atggacaagg agctcaagga tatggaacag
gaagtggagc taccatcgct 840ttaactgctg gtggacttgg aggttatggt ggacagagag
gtcaagttgg tcaaggacca 900tcaggacaat tagctggagc acctggtgga tatggacaag
gaagtgcagc cgccacggca 960gctggtggac ttcgaggttt cgggcaaggg ttacaagtac
10002306PRTNephila pilipes 2Gly Gly Leu Gly Gly Ser
Gly Gly Gln Gly Gly Gln Ile Pro Ser Gly 1 5
10 15 Ala Val Gly Gln Gly Thr Gln Gly Tyr Gly Thr
Gly Ser Gly Ala Thr 20 25
30 Ile Ala Leu Thr Ala Gly Gly Leu Gly Gly Gln Gly Gly Gln Gly
Pro 35 40 45 Ser
Gly Ser Gly Gly Gln Gly Pro Ser Gly Gln Gly Ala Gln Gly Pro 50
55 60 Gly Gly Tyr Gly Ala Gly
Asn Ala Ala Ala Ala Asn Ala Ala Ala Ser 65 70
75 80 Gly Leu Gly Gly Tyr Gly Val Gly Gly Gln Gly
Ser Gly Gln Arg Pro 85 90
95 Ser Gly Ala Gly Gly Gln Gly Ala Gln Ala Pro Gly Gly Tyr Gly Thr
100 105 110 Gly Ser
Gly Ser Thr Ile Val Ile Thr Ala Gly Gly Gln Arg Gly Gln 115
120 125 Gly Gly Gln Gly Pro Ser Gly
Gln Leu Ala Gln Ala Pro Ser Gly Tyr 130 135
140 Gly Gln Gly Ser Gly Ala Ala Ala Ala Ser Gly Gly
Leu Gly Gly Tyr 145 150 155
160 Gly Gly Gln Gly Gly Gln Arg Ser Ser Gly Ala Gly Ala Gln Gly Thr
165 170 175 Gln Gly Tyr
Gly Thr Gly Ser Gly Thr Thr Ile Ala Leu Thr Ala Gly 180
185 190 Gly Ile Gly Gly Ser Gly Gly Gln
Gly Gly Gln Arg Pro Ser Gly Ile 195 200
205 Gly Gly Gln Gly Ala Gln Gly Pro Gly Gly Tyr Gly Ala
Gly Ser Gly 210 215 220
Ser Thr Ile Ala Ile Thr Thr Gly Gly Leu Gly Gly Tyr Gly Gly Gln 225
230 235 240 Gly Gly Gln Arg
Pro Ser Gly Ala Asp Gly Gln Gly Ala Gln Gly Tyr 245
250 255 Gly Thr Gly Ser Gly Ala Thr Ile Ala
Leu Thr Ala Gly Gly Leu Gly 260 265
270 Gly Tyr Gly Gly Gln Arg Gly Gln Val Gly Gln Gly Pro Ser
Gly Gln 275 280 285
Leu Ala Gly Ala Pro Gly Gly Tyr Gly Gln Gly Ser Ala Ala Ala Thr 290
295 300 Ala Ala 305
3111DNANephila pilipes 3ggtggtcttg gtggatctgg aggtcaaggt ggccaaatac
catctggcgc tgttggacaa 60ggaactcaag gatatggaac aggaagtgga gcaaccatcg
cattaactgc t 111437PRTNephila pilipes 4Gly Gly Leu Gly Gly
Ser Gly Gly Gln Gly Gly Gln Ile Pro Ser Gly 1 5
10 15 Ala Val Gly Gln Gly Thr Gln Gly Tyr Gly
Thr Gly Ser Gly Ala Thr 20 25
30 Ile Ala Leu Thr Ala 35 5126DNANephila
pilipes 5ggtggacttg ggggacaagg tggtcaagga ccatctggct ctggtggaca
aggcccatca 60ggacaaggag ctcaaggacc tggcggatat ggagcaggaa atgcagccgc
cgccaacgca 120gcagct
126642PRTNephila pilipes 6Gly Gly Leu Gly Gly Gln Gly Gly Gln
Gly Pro Ser Gly Ser Gly Gly 1 5 10
15 Gln Gly Pro Ser Gly Gln Gly Ala Gln Gly Pro Gly Gly Tyr
Gly Ala 20 25 30
Gly Asn Ala Ala Ala Ala Asn Ala Ala Ala 35 40
7129DNANephila pilipes 7agtggacttg gaggctatgg agttggtggg cagggaagtg
gccaaagacc atctggagct 60ggtggacaag gtgctcaagc gccaggtgga tatggaacag
gaagtggatc gaccatcgta 120ataactgct
129843PRTNephila pilipes 8Ser Gly Leu Gly Gly Tyr
Gly Val Gly Gly Gln Gly Ser Gly Gln Arg 1 5
10 15 Pro Ser Gly Ala Gly Gly Gln Gly Ala Gln Ala
Pro Gly Gly Tyr Gly 20 25
30 Thr Gly Ser Gly Ser Thr Ile Val Ile Thr Ala 35
40 996DNANephila pilipes 9ggtggacaga gaggacaagg
tggtcaagga ccatcaggac aattagctca agcacctagt 60ggatatggac aaggaagtgg
agccgccgcc gcctct 961032PRTNephila pilipes
10Gly Gly Gln Arg Gly Gln Gly Gly Gln Gly Pro Ser Gly Gln Leu Ala 1
5 10 15 Gln Ala Pro Ser
Gly Tyr Gly Gln Gly Ser Gly Ala Ala Ala Ala Ser 20
25 30 11111DNANephila pilipes
11ggtggtcttg gaggatatgg aggtcaaggt ggccaaagat catctggcgc tggtgcacaa
60ggaactcaag gatatggtac aggaagtgga acaactatcg cattaactgc t
1111237PRTNephila pilipes 12Gly Gly Leu Gly Gly Tyr Gly Gly Gln Gly Gly
Gln Arg Ser Ser Gly 1 5 10
15 Ala Gly Ala Gln Gly Thr Gln Gly Tyr Gly Thr Gly Ser Gly Thr Thr
20 25 30 Ile Ala
Leu Thr Ala 35 13120DNANephila pilipes 13ggtggtattg
gaggatctgg aggtcaaggt ggccaaagac catctggcat tggtggacaa 60ggagctcaag
ggccaggtgg atatggagca ggaagtggat ctaccatcgc aataactact
1201440PRTNephila pilipes 14Gly Gly Ile Gly Gly Ser Gly Gly Gln Gly Gly
Gln Arg Pro Ser Gly 1 5 10
15 Ile Gly Gly Gln Gly Ala Gln Gly Pro Gly Gly Tyr Gly Ala Gly Ser
20 25 30 Gly Ser
Thr Ile Ala Ile Thr Thr 35 40 15111DNANephila
pilipes 15ggtggtcttg gtggatatgg aggtcaaggt ggccaaagac catctggcgc
tgatggacaa 60ggagctcaag gatatggaac aggaagtgga gctaccatcg ctttaactgc t
1111637PRTNephila pilipes 16Gly Gly Leu Gly Gly Tyr Gly Gly
Gln Gly Gly Gln Arg Pro Ser Gly 1 5 10
15 Ala Asp Gly Gln Gly Ala Gln Gly Tyr Gly Thr Gly Ser
Gly Ala Thr 20 25 30
Ile Ala Leu Thr Ala 35 17114DNANephila pilipes
17ggtggacttg gaggttatgg tggacagaga ggtcaagttg gtcaaggacc atcaggacaa
60ttagctggag cacctggtgg atatggacaa ggaagtgcag ccgccacggc agct
1141838PRTNephila pilipes 18Gly Gly Leu Gly Gly Tyr Gly Gly Gln Arg Gly
Gln Val Gly Gln Gly 1 5 10
15 Pro Ser Gly Gln Leu Ala Gly Ala Pro Gly Gly Tyr Gly Gln Gly Ser
20 25 30 Ala Ala
Ala Thr Ala Ala 35 19900DNANephila pilipes
19ggtggtcttg gtggatatgg aggtcaaggt ggccaaagac tatctggcgc tagtggacaa
60ggaactcaag gatatggaac aggaagtgga gctaccatcg ctttaactgc tggtggtcaa
120ggtggatctg gaggtcaagg tggccaaaga ctatctggcg ctagtggaca aggaactcaa
180ggatatggaa caggaagtgg agcaaccatc gcattaactg ctggtggact tgggggacaa
240ggtggtcaag gaccatctgg ctctggtgga caaggcccat caggacaagg agctcaaggg
300ccaggtggat atggaacagg aagtggaacg gccatcgcaa taactgctgg tggacagaga
360ggacaaggtg gtcaaggacc atcaggacaa ttagctcaag cacctagtgg atatggacta
420ggaagtggag ccgccgccgc ctttggtggt cttggaggat atggaggtca aggtggccaa
480agatcatctg gcgctggtgc acaaggaact caaggatatg gaacaggaag tggaacaact
540atcgcattaa ctgctggtgg tattggagga tctggaggtc aaggtggcca aagaccatct
600ggcgctggtg gacaaggagc tcaagggcca ggtggatatg gagcaggaag tggatctacc
660atcgcaataa cagctggtgg tcttggtgga tctggaggtc aaggtggcca aagaccatct
720ggcgctggtg gacaaggagc tcaaggatat ggaacaggaa gtggagctac catcgcaata
780actgctggtg gacttggagg ttatggtgga cagagaggtc aagttggtca aggaccatca
840ggacaattag ctggagcacc tggtggatat ggacaaggaa gtgcagccgc cacggcagct
90020300PRTNephila pilipes 20Gly Gly Leu Gly Gly Tyr Gly Gly Gln Gly Gly
Gln Arg Leu Ser Gly 1 5 10
15 Ala Ser Gly Gln Gly Thr Gln Gly Tyr Gly Thr Gly Ser Gly Ala Thr
20 25 30 Ile Ala
Leu Thr Ala Gly Gly Gln Gly Gly Ser Gly Gly Gln Gly Gly 35
40 45 Gln Arg Leu Ser Gly Ala Ser
Gly Gln Gly Thr Gln Gly Tyr Gly Thr 50 55
60 Gly Ser Gly Ala Thr Ile Ala Leu Thr Ala Gly Gly
Leu Gly Gly Gln 65 70 75
80 Gly Gly Gln Gly Pro Ser Gly Ser Gly Gly Gln Gly Pro Ser Gly Gln
85 90 95 Gly Ala Gln
Gly Pro Gly Gly Tyr Gly Thr Gly Ser Gly Thr Ala Ile 100
105 110 Ala Ile Thr Ala Gly Gly Gln Arg
Gly Gln Gly Gly Gln Gly Pro Ser 115 120
125 Gly Gln Leu Ala Gln Ala Pro Ser Gly Tyr Gly Leu Gly
Ser Gly Ala 130 135 140
Ala Ala Ala Phe Gly Gly Leu Gly Gly Tyr Gly Gly Gln Gly Gly Gln 145
150 155 160 Arg Ser Ser Gly
Ala Gly Ala Gln Gly Thr Gln Gly Tyr Gly Thr Gly 165
170 175 Ser Gly Thr Thr Ile Ala Leu Thr Ala
Gly Gly Ile Gly Gly Ser Gly 180 185
190 Gly Gln Gly Gly Gln Arg Pro Ser Gly Ala Gly Gly Gln Gly
Ala Gln 195 200 205
Gly Pro Gly Gly Tyr Gly Ala Gly Ser Gly Ser Thr Ile Ala Ile Thr 210
215 220 Ala Gly Gly Leu Gly
Gly Ser Gly Gly Gln Gly Gly Gln Arg Pro Ser 225 230
235 240 Gly Ala Gly Gly Gln Gly Ala Gln Gly Tyr
Gly Thr Gly Ser Gly Ala 245 250
255 Thr Ile Ala Ile Thr Ala Gly Gly Leu Gly Gly Tyr Gly Gly Gln
Arg 260 265 270 Gly
Gln Val Gly Gln Gly Pro Ser Gly Gln Leu Ala Gly Ala Pro Gly 275
280 285 Gly Tyr Gly Gln Gly Ser
Ala Ala Ala Thr Ala Ala 290 295 300
21111DNANephila pilipes 21ggtggtcttg gtggatatgg aggtcaaggt ggccaaagac
tatctggcgc tagtggacaa 60ggaactcaag gatatggaac aggaagtgga gctaccatcg
ctttaactgc t 1112237PRTNephila pilipes 22Gly Gly Leu Gly Gly
Tyr Gly Gly Gln Gly Gly Gln Arg Leu Ser Gly 1 5
10 15 Ala Ser Gly Gln Gly Thr Gln Gly Tyr Gly
Thr Gly Ser Gly Ala Thr 20 25
30 Ile Ala Leu Thr Ala 35 23111DNANephila
pilipes 23ggtggtcaag gtggatctgg aggtcaaggt ggccaaagac tatctggcgc
tagtggacaa 60ggaactcaag gatatggaac aggaagtgga gcaaccatcg cattaactgc t
1112437PRTNephila pilipes 24Gly Gly Gln Gly Gly Ser Gly Gly
Gln Gly Gly Gln Arg Leu Ser Gly 1 5 10
15 Ala Ser Gly Gln Gly Thr Gln Gly Tyr Gly Thr Gly Ser
Gly Ala Thr 20 25 30
Ile Ala Leu Thr Ala 35 25126DNANephila pilipes
25ggtggacttg ggggacaagg tggtcaagga ccatctggct ctggtggaca aggcccatca
60ggacaaggag ctcaagggcc aggtggatat ggaacaggaa gtggaacggc catcgcaata
120actgct
1262642PRTNephila pilipes 26Gly Gly Leu Gly Gly Gln Gly Gly Gln Gly Pro
Ser Gly Ser Gly Gly 1 5 10
15 Gln Gly Pro Ser Gly Gln Gly Ala Gln Gly Pro Gly Gly Tyr Gly Thr
20 25 30 Gly Ser
Gly Thr Ala Ile Ala Ile Thr Ala 35 40
2796DNANephila pilipes 27ggtggacaga gaggacaagg tggtcaagga ccatcaggac
aattagctca agcacctagt 60ggatatggac taggaagtgg agccgccgcc gccttt
962832PRTNephila pilipes 28Gly Gly Gln Arg Gly
Gln Gly Gly Gln Gly Pro Ser Gly Gln Leu Ala 1 5
10 15 Gln Ala Pro Ser Gly Tyr Gly Leu Gly Ser
Gly Ala Ala Ala Ala Phe 20 25
30 29111DNANephila pilipes 29ggtggtcttg gaggatatgg aggtcaaggt
ggccaaagat catctggcgc tggtgcacaa 60ggaactcaag gatatggaac aggaagtgga
acaactatcg cattaactgc t 1113037PRTNephila pilipes 30Gly Gly
Leu Gly Gly Tyr Gly Gly Gln Gly Gly Gln Arg Ser Ser Gly 1 5
10 15 Ala Gly Ala Gln Gly Thr Gln
Gly Tyr Gly Thr Gly Ser Gly Thr Thr 20 25
30 Ile Ala Leu Thr Ala 35
31120DNANephila pilipes 31ggtggtattg gaggatctgg aggtcaaggt ggccaaagac
catctggcgc tggtggacaa 60ggagctcaag ggccaggtgg atatggagca ggaagtggat
ctaccatcgc aataacagct 1203240PRTNephila pilipes 32Gly Gly Ile Gly Gly
Ser Gly Gly Gln Gly Gly Gln Arg Pro Ser Gly 1 5
10 15 Ala Gly Gly Gln Gly Ala Gln Gly Pro Gly
Gly Tyr Gly Ala Gly Ser 20 25
30 Gly Ser Thr Ile Ala Ile Thr Ala 35
40 33111DNANephila pilipes 33ggtggtcttg gtggatctgg aggtcaaggt ggccaaagac
catctggcgc tggtggacaa 60ggagctcaag gatatggaac aggaagtgga gctaccatcg
caataactgc t 1113437PRTNephila pilipes 34Gly Gly Leu Gly Gly
Ser Gly Gly Gln Gly Gly Gln Arg Pro Ser Gly 1 5
10 15 Ala Gly Gly Gln Gly Ala Gln Gly Tyr Gly
Thr Gly Ser Gly Ala Thr 20 25
30 Ile Ala Ile Thr Ala 35 35114DNANephila
pilipes 35ggtggacttg gaggttatgg tggacagaga ggtcaagttg gtcaaggacc
atcaggacaa 60ttagctggag cacctggtgg atatggacaa ggaagtgcag ccgccacggc
agct 1143638PRTNephila pilipes 36Gly Gly Leu Gly Gly Tyr Gly Gly
Gln Arg Gly Gln Val Gly Gln Gly 1 5 10
15 Pro Ser Gly Gln Leu Ala Gly Ala Pro Gly Gly Tyr Gly
Gln Gly Ser 20 25 30
Ala Ala Ala Thr Ala Ala 35 3712PRTNephila pilipes
37Gly Gly Leu Gly Gly Ser Gly Gly Gln Gly Gly Gln 1 5
10 389PRTNephila pilipes 38Gly Gly Leu Gly Gly Gln
Gly Gly Gln 1 5 3915PRTNephila pilipes
39Ser Gly Leu Gly Gly Tyr Gly Val Gly Gly Gln Gly Ser Gly Gln 1
5 10 15 409PRTNephila pilipes
40Gly Gly Gln Arg Gly Gln Gly Gly Gln 1 5
4112PRTNephila pilipes 41Gly Gly Leu Gly Gly Tyr Gly Gly Gln Gly Gly Gln
1 5 10 4212PRTNephila pilipes
42Gly Gly Ile Gly Gly Ser Gly Gly Gln Gly Gly Gln 1 5
10 4312PRTNephila pilipes 43Gly Gly Leu Gly Gly Tyr
Gly Gly Gln Gly Gly Gln 1 5 10
4415PRTNephila pilipes 44Gly Gly Leu Gly Gly Tyr Gly Gly Gln Arg Gly Gln
Val Gly Gln 1 5 10 15
4512PRTNephila pilipes 45Gly Gly Gln Gly Gly Ser Gly Gly Gln Gly Gly Gln
1 5 10 465PRTNephila pilipes
46Ile Pro Ser Gly Ala 1 5 475PRTNephila pilipes 47Gly Pro
Ser Gly Ser 1 5 485PRTNephila pilipes 48Arg Pro Ser Gly
Ala 1 5 495PRTNephila pilipes 49Gly Pro Ser Gly Gln 1
5 505PRTNephila pilipes 50Arg Ser Ser Gly Ala 1
5 515PRTNephila pilipes 51Arg Pro Ser Gly Ile 1 5
525PRTNephila pilipes 52Arg Leu Ser Gly Ala 1 5
533PRTNephila pilipes 53Val Gly Gln 1 543PRTNephila pilipes
54Gly Gly Gln 1 553PRTNephila pilipes 55Leu Ala Gln 1
563PRTNephila pilipes 56Gly Ala Gln 1 573PRTNephila pilipes
57Asp Gly Gln 1 583PRTNephila pilipes 58Leu Ala Gly 1
593PRTNephila pilipes 59Ser Gly Gln 1 603PRTNephila pilipes
60Gly Thr Gln 1 613PRTNephila pilipes 61Gly Ala Gln 1
623PRTNephila pilipes 62Gly Pro Gly 1 633PRTNephila pilipes
63Ala Pro Gly 1 643PRTNephila pilipes 64Ala Pro Ser 1
657PRTNephila pilipes 65Gly Tyr Gly Thr Gly Ser Gly 1 5
665PRTNephila pilipes 66Gly Tyr Gly Ala Gly 1 5
677PRTNephila pilipes 67Gly Tyr Gly Gln Gly Ser Gly 1 5
687PRTNephila pilipes 68Gly Tyr Gly Ala Gly Ser Gly 1
5 696PRTNephila pilipes 69Gly Tyr Gly Gln Gly Ser 1
5 707PRTNephila pilipes 70Gly Tyr Gly Leu Gly Ser Gly 1
5 717PRTNephila pilipes 71Ala Thr Ile Ala Leu Thr Ala 1
5 727PRTNephila pilipes 72Ser Thr Ile Val Ile Thr Ala
1 5 737PRTNephila pilipes 73Thr Thr Ile Ala Leu
Thr Ala 1 5 747PRTNephila pilipes 74Ser Thr Ile
Ala Ile Thr Thr 1 5 757PRTNephila pilipes 75Thr
Ala Ile Ala Ile Thr Ala 1 5 767PRTNephila pilipes
76Ser Thr Ile Ala Ile Thr Ala 1 5 777PRTNephila
pilipes 77Ala Thr Ile Ala Ile Thr Ala 1 5
789PRTNephila pilipes 78Asn Ala Ala Ala Ala Asn Ala Ala Ala 1
5 795PRTNephila pilipes 79Ala Ala Ala Ala Ser 1
5 806PRTNephila pilipes 80Ala Ala Ala Thr Ala Ala 1
5 815PRTNephila pilipes 81Ala Ala Ala Ala Phe 1 5
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