# Patent

## Title
Nucleic acid encoding a silk protein and a method of producing a silk thread using same

## Abstract

Disclosed are a protein encoded by any one of nucleic acids (i) to (iv): (i) a nucleic acid having a base sequence of SEQ ID NO: 1; (ii) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 2; (iii) a nucleic acid encoding a dragline protein and having a sequence identity of 90% or more with the nucleic acid (i); (iv) a nucleic acid which encodes a dragline protein and hybridizes with a complementary chain of the nucleic acid (i) under stringent conditions, and a silk thread containing the protein.

## Background
This application is a divisional of U.S. patent application Ser. No. 13/524,120 filed Jun. 15, 2012 (now allowed), which is a divisional of U.S. patent application Ser. No. 13/226,964 filed Sep. 7, 2011 (now U.S. Pat. No. 8,222,479) which claims the benefit of Japanese Patent Application No. 2010-203558, filed on Sep. 10, 2010, in the Japanese Patent Office, the disclosures of which are incorporated herein in their entireties by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a recombinant organism and a protein produced by the recombinant organism.
2. Related Background Art
As a natural fiber with an excellent strength, spider silk has drawn an attention. However, spiders eat each other and thus are not suitable for farming in a same place, and the amount of spider silk obtained from a single spider is low. In addition, a spider uses different types of silk threads according to its needs. Therefore, a mass production of spider silk is difficult.
Then, an attempt has been made to introduce a gene encoding a spider silk protein into an organism excluding a spider by use of a gene recombinant technique to produce spider silk. For example, a method of producing a spider silk protein in a genetically modified goat and obtaining the spider silk protein from milk of the goat is disclosed (Patent Literature 1: Japanese Unexamined Patent Application Publication No. 2002-506642). However, according to the method, the spider silk protein needs to be extracted, purified and artificially spun, being problematic in the points of labor, cost and environmental load due to a solvent.
To solve the aforementioned problems, an attempt has been made to introduce a gene encoding a spider silk protein into a silkworm to produce the spider silk protein (Patent Literature 2: WO2005/068495). By a recombinant silkworm, the spider silk protein is ejected as a silk thread, and therefore the aforementioned treatments such as extraction, purification and spinning are unnecessary.
SUMMARY OF THE INVENTION
However, physical properties of silk threads produced by conventional recombinant silkworms have been far inferior to those of native spider silks, and the silk threads have been unsatisfactory in their strength.
Then, the present invention aims to provide a recombinant organism producing a protein with excellent physical properties and a protein with excellent physical properties produced by the recombinant organism. Furthermore, the present invention aims to provide a recombinant silkworm producing a silk thread with a sufficiently excellent strength and a silk thread with sufficiently excellent strength produced by the recombinant silkworm.
The present inventors intensively studied with a view to achieving the aforementioned aims, and, as a result, found that when a gene of Argiope bruennichi is introduced into an organism to be genetically modified, the resultant protein has excellent physical properties, and thereby completed the present invention.
More specifically, the present invention relates to a recombinant organism having any one of the following nucleic acids (i) to (iv) introduced therein and a protein produced by the recombinant organism:
(i) a nucleic acid having a base sequence of SEQ ID NO: 1;
(ii) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 2;
(iii) a nucleic acid encoding a dragline protein and having a sequence identity of 90% or more with the nucleic acid (i);
(iv) a nucleic acid which encodes a dragline protein and hybridizes with a complementary chain of the nucleic acid (i) under stringent conditions.
By introducing the aforementioned specific nucleic acid, a protein produced by the recombinant organism becomes rich in the spider protein and, as a result, becomes excellent in physical properties such as strength.
Particularly, the present invention relates to a recombinant silkworm having any one of the aforementioned nucleic acids (i) to (iv) introduced therein, a protein produced by the recombinant silkworm and a silk thread produced by the recombinant silkworm. By introducing the aforementioned specific nucleic acid into a silkworm, a silk thread produced by the recombinant silkworm is rich in spider protein and, as a result, has a sufficient strength.
According to the present invention, it is possible to provide a recombinant organism which produces a protein with excellent physical properties, and a protein with excellent physical properties produced by the recombinant organism. Furthermore, according to in an embodiment, it is possible to provide a recombinant silkworm which produces a silk thread with a sufficiently excellent strength and a silk thread with a sufficiently excellent strength produced by the recombinant silkworm.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic view showing the structure of a vector plasmid for genetic recombination. In FIG. 1, legends have the following meanings: FP: Silkworm fibroin H-chain gene promoter; MASP: Spider gene; FC: C-terminal partial sequence of the silkworm fibroin H-chain gene; MK: Marker gene; L: PiggyBac transposon L-hand; and R: PiggyBac transposon R-hand.
FIG. 2 is a view showing a genome sequence of a silkworm downstream of the site at which a spider gene expression cassette is inserted.
FIG. 3 is a view showing the result of the SDS-PAGE separation of silk thread proteins produced by a recombinant silkworm.
FIG. 4 shows scanning electron micrographs of silk threads of a recombinant silkworm (A) and a non-recombinant silkworm (B).



DESCRIPTION OF THE PREFERRED EMBODIMENTS
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.
The present invention relates to a recombinant organism having any one of the following nucleic acids (i) to (iv) introduced therein and a protein produced by the recombinant organism:
(i) a nucleic acid having a base sequence of SEQ ID NO: 1;
(ii) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 2;
(iii) a nucleic acid encoding a dragline protein and having a sequence identity of 90% or more with the nucleic acid (i);
(iv) a nucleic acid which encodes a dragline protein and hybridizes with a complementary chain of the nucleic acid (i) under stringent conditions.
Particularly, the present invention relates to a recombinant silkworm having any one of the aforementioned nucleic acids (i) to (iv) introduced therein, a protein produced by the recombinant silkworm and a silk thread produced by the recombinant silkworm.
A nucleic acid having the base sequence of SEQ ID NO: 1 is a nucleic acid encoding MaSp1 (major ampullate spidroin 1) protein, which is a main component of a dragline (or warp) protein of Argiope bruennichi, 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 a restriction enzyme(s), as long as a nucleic acid has the base sequence of SEQ ID NO: 1.
The amino acid sequence of SEQ ID NO: 2 is the amino acid sequence of the MaSp1 protein of Argiope bruennichi. 
As the nucleic acid to be introduced into a silkworm, a nucleic acid (iii) having a sequence identity of 90% or more with the nucleic acid having a base sequence of SEQ ID NO: 1 may be used as long as it encodes a dragline protein. The sequence identity may be 90% or more, but is preferably 95% or more and more preferably 98% or more.
Furthermore, the nucleic acid to be introduced into a silkworm may be a nucleic acid (iv) which hybridizes with a complementary chain of a nucleic acid having the base sequence of SEQ ID NO: 1 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 a complementary bonding between complementary chains or form interaction between bases of single-strand nucleic acid molecules.
“Stringent conditions” mentioned above 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, N.Y.).
Typically, the stringent conditions are those in which the salt concentration is less than about 1.0 M Na+, typically about 0.01 to 1.0 M of Na+ 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 a destabilizing 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.
In this specification, the term “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.
In this specification, the word “silkworm” refers to Bombyx mori. A silkworm may be either a breed for experimentation or a commercial breed commercialized for practical use. Furthermore, the word “recombinant silkworm” refers to a silkworm transformed by introducing a foreign gene into the silkworm chromosome by means of genetic recombination. In an embodiment, genetic recombination is performed by a method 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.
In this specification, the word “silk thread” is a fiber, which is ejected by Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, etc., constituting a cocoon and containing a fibroin protein as a main component. The fibroin protein is composed of two large and small subunits (H-chain and L chain).
Examples
The present invention will be more specifically described by way of Examples. However, the present invention is not limited to the following Examples.
(Spider Gene)
A spider gene was obtained in accordance with a PCR method by using a vector containing a nucleic acid having the base sequence of SEQ ID NO: 1 and primers designed so as to match respectively with two ends of the nucleic acid. To the primers, appropriate restriction enzyme sites are previously provided for the following gene manipulation. More specifically, as a forward primer, MaSp1FW (5′-CGACTCACTATAGGGAATTCCTTAACTAGTGGAGCAGCC-3′) (SEQ ID NO: 3) was used and as a reverse primer, MaSp1RV (5′-GACAATCCGTATACCAAGCTTTCTCTGCTAGCTAG-3′) (SEQ ID NO: 4) was used.
(Silkworm Fibroin H-Chain Gene Promoter Sequence)
A silkworm fibroin H-chain gene promoter sequence was obtained in accordance with a PCR method by using primers designed based on the sequence of the silkworm fibroin H-chain gene (GeneBank Registration No. AF226688) and normal silkworm genomic DNA as a template. More specifically, as a forward primer, PfibH5′ (5′-AAGCTTGTTGTACAAAACTGCC-3′) (SEQ ID NO: 5) containing a HindIII restriction enzyme site was used and as a reverse primer, PfibH3′ (5′-TGCAGCACTAGTGCTGAAATCGCT-3′) (SEQ ID NO: 6) containing a SpeI site was used.
(C-Terminal Partial Sequence of Silkworm Fibroin H-Chain Gene)
A C-terminal partial sequence of the silkworm fibroin H-chain gene was obtained in accordance with a PCR method by using primers designed based on the sequence of the silkworm fibroin H-chain gene (GeneBank Registration No. AF226688) and normal silkworm genomic DNA as a template. More specifically, as a forward primer, LBS-FW (5′-CTAGCTAGCAGTTACGGAGCTGGCAGGG-3′) (SEQ ID NO: 7) containing a NheI site was used and as a reverse primer, LBS-RV (5′-CGGGATCCTAGTACATTCAAATAAAATGCATAC-3′) (SEQ ID NO: 8) containing a BamHI site was used.
(Preparation of Vector Plasmid for Genetic Recombination)
The silkworm fibroin H-chain promoter sequence (FP), a spider gene sequence (MASP) and the C-terminal partial sequence of the silkworm fibroin H-chain gene (FC) were sequentially ligated to form a spider gene expression cassette. The spider gene expression cassette was introduced into a vector plasmid containing piggyBac transposon to prepare a vector plasmid for genetic recombination. FIG. 1 is a schematic view showing the structure of the vector plasmid for genetic recombination. The reference symbols of FIG. 1 stand for the followings.
FP: Silkworm fibroin H-chain gene promoter sequence
MASP: Spider gene sequence
FC: C-terminal partial sequence of the silkworm fibroin H-chain gene
MK: Marker gene sequence
L: PiggyBac transposon L-hand
R: PiggyBac transposon R-hand
(Preparation of Recombinant Silkworm)
The vector plasmid for genetic recombination was amplified in Escherichia coli, and purified by “QIAGEN plasmid Midi Kit” (manufactured by QIAGEN) in accordance with the manual attached the kit. A helper plasmid containing a transposase protein gene was also purified by the above method. DNA of the plasmids purified above were dissolved in TE buffer such that the DNA of the vector plasmid for genetic recombination and the DNA of the helper plasmid were mixed in a ratio of 1:1 and precipitated with ethanol. Finally, the concentration of the DNA mixture was adjusted with a phosphate buffer (pH 7) containing 5 mM KCl to be 400 ng/ul. This was injected to a silkworm egg of 3-6 hour old after egg-laying under microscopic observation.
The egg to which the sample was injected is designated as G0 generation; the G0 generation grew and changed to a moth, which is designated as G0 moth; eggs laid by G0 moth as a parent are designated as G1 eggs. G0 moth was mated with a moth having no injection and G1 eggs were collected; and G1 eggs in the incubation state were observed under a fluorescent microscope and screened for fluorescence-emitting eggs (positive egg). A population of eggs laid by a single parent moth (G0 moth) was treated as a group and the number of groups in which one or more positive eggs were detected was counted and recorded as the “positive G1 number”. The results are shown in Table 1.










TABLE 1





Number of
Number of 

Number of


injection eggs
hatched eggs
Hatching rate
Positive G1 (moth)







6426
1200
18.7
12









(Insertion of Spider Gene by Inverse PCR and Confirmation of Insertion Site)
Silkworm genomic DNA was extracted by a known method (see Sambrook and Maniatis, Molecular Cloning—A Laboratory Manual). The genomic DNA was cleaved with restriction enzyme HaeIII and thereafter self-ligated. Using this as a template and two pairs of primers respectively matching with left and right hands of piggyBac, and a fragment containing a silkworm genome sequence of an insertion site was amplified to identify the sequence. More specifically, as a left hand forward primer, BacLF (5′-CTTGACCTTGCCACAGAGGACTATTAGAGG-3′) (SEQ ID NO: 9) was used, as a left hand reverse primer, BacLR (5′-CAGTGACACTTACCGCATTGACAAGCACGC-3′) (SEQ ID NO: 10) was used, as a right hand forward primer, (5′-CCTCGATATACAGACCGATAAAACACATG-3′) (SEQ ID NO: 11) was used, and a right hand reverse primer, (5′-GTCAGTCAGAAACAACTTTGGCACATATC-3′) (SEQ ID NO: 12) was used. FIG. 2 shows a silkworm genome sequence (SEQ ID NO: 13) downstream of a site at which a spider gene expression cassette is inserted.
(Protein Analysis of Silk Thread)
A cocoon produced by a recombinant silkworm was excised, and to 1 mg of cocoon pieces, 50 μl of a 60% LiSCN was added, which was shaken, allowed to stand still at room temperature for 2 hours to solubilize a protein, thereafter, centrifuged at 15000 rpm to remove an unsolved substances, and the supernatant was subjected to SDS-PAGE to separate a spider silk protein and fibroin of a silk worm. FIG. 3 shows the SDS-PAGE separation results of the silk thread protein produced by a recombinant silkworm. In FIG. 3, FH stands for a silkworm fibroin H-chain and MASP stands for a spider silk protein. Furthermore, in FIG. 3, in lane 1, an HMW molecular weight marker was run; in lane 2, the supernatant containing a silk thread protein produced by a recombinant silkworm was run; in lane 3, a solution containing 5 times more protein than that in lane 2 was run. From the separation results, the content of the spider silk protein in the silk thread protein was calculated to be 22.5%, which is a numerical value significantly higher than the content of a conventional recombinant silkworm.
(Determination of Fineness of Silk Thread Under Scanning Electron Microscope Observation)
A cocoon produced by a recombinant silkworm was soaked in warm water of 40° C. for one minute, fluff was removed, and thread was carefully collected and acclimated at conditions: 20° C., 65% RH, a day and night to prepare a sample. The obtained sample was observed by SEM (scanning electron microscope) S-2380N (manufactured by Hitachi, Ltd.) at a voltage of 10 kV and photographed. Based on the photograph, the diameter and sectional area of a thread were estimated, and based on the sectional area, the strength of the thread was calculated. As a control, a cocoon of a silkworm (non-recombinant silkworm) not genetically modified was treated in the same manner. FIG. 4 shows scanning electron micrographs of silk threads of a recombinant silkworm (A) and a non-recombinant silkworm (B).
(Analysis of Physical Property of Silk Thread)
The cocoon produced by a recombinant silkworm was soaked in warm water of 40° C. for one minute, fluff was removed, and thread was carefully collected and acclimated at conditions: 20° C., 65% RH, a day and night to prepare a sample. The obtained sample was tested for tensile strength by use of “AUTOGRAPH AGS-J” (manufactured by Shimadzu Corporation) under standard conditions: 20° C., 65% RH. As a control, the silk thread produced by a silkworm (non-recombinant silkworm) not genetically modified was tested for tensile strength in the same manner. As a result, the strength of the silk thread of a control (non-recombinant silkworm) was 397.0 MPa (3.49 g/d), whereas the strength of the silk thread produced by the recombinant silkworm was 489.2 MPa (4.27 g/d), which was increased by 22.35%.
On the other hand, in a conventional recombinant silkworm employing a gene of e.g., Araneus ventricosus described in Patent Literature 2, the strength of a control silk thread was 3.78 g/d, whereas the strength of the silk thread of a recombinant silkworm was 3.93 g/d, which was increased only by 3.96% (see Patent Literature 2, page 29, Table 3).
From the foregoing, in the case of using a specific nucleic acid of the present invention, compared to a conventional case of using a gene of e.g. Araneus ventricosus, the strength of silk threads produced by the recombinant silkworms was found to clearly increase. Table 2 shows physical properties of the silk thread of the present invention compared to those of the silk thread of Patent Literature 2.











TABLE 2






Non-






recombinant
Recombinant
Increased rate
Content of



silkworm
silkworm
of strength
spider-thread



thread (g/d)
thread (g/d)
(%)
protein (%)



















Patent
3.78
3.93
3.96
5


Literature 2






Present
3.49
4.27
22.35
22.5


Invention









A protein produced by the recombinant organism of the present invention is useful as a naturally occurring material with excellent physical properties. Particularly, the silk thread produced by the recombinant silkworm of the present invention is a naturally occurring material with a sufficiently excellent strength, and thus, the silk thread is preferably used not only in medical supplies such as surgical suture but also various uses such as flight equipment, clothing and cosmetics.

## Claims

1. An isolated protein encoded by a nucleic acid selected from the group consisting of:
(i) a nucleic acid having the base sequence of SEQ ID NO:1;
(ii) a nucleic acid encoding a protein having the amino acid sequence of SEQ ID NO: 2;
(iii) a nucleic acid with a base sequence identity of 90% or more to the nucleic acid sequence of clause (i); and
(iv) a nucleic acid which encodes a dragline protein and hybridizes with the complementary chain of the nucleic acid of clause (i) under stringent conditions comprising hybridization in a buffer solution of 50% formamide, 1M NaCl, 1% SDS at 37° C. and washing with 0.1×SSC at 60° C.

2. A silk thread comprising the protein according to claim 1.