WO2011112046A2 - 고분자량의 재조합 실크 또는 실크 유사 단백질 및 이를 이용하여 제조된 마이크로 또는 나노 크기의 거미줄 또는 거미줄 유사 섬유 - Google Patents
고분자량의 재조합 실크 또는 실크 유사 단백질 및 이를 이용하여 제조된 마이크로 또는 나노 크기의 거미줄 또는 거미줄 유사 섬유 Download PDFInfo
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- WO2011112046A2 WO2011112046A2 PCT/KR2011/001730 KR2011001730W WO2011112046A2 WO 2011112046 A2 WO2011112046 A2 WO 2011112046A2 KR 2011001730 W KR2011001730 W KR 2011001730W WO 2011112046 A2 WO2011112046 A2 WO 2011112046A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/06—Wet spinning methods
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
- D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
Definitions
- the present invention relates to a high molecular weight recombinant silk or silk-like protein having a molecular weight almost similar to that of natural silk protein, and micro or nano-sized spider web or spider web-like fiber having improved physical properties.
- Dragline spider web used as spider's lifeline and used to make radial spider webs, has great strength and elasticity. Cobwebs are five times stronger per unit mass than steel and three times harder than man-made high quality Kepler fibers (Gosline, JM et al., J. Exp. Biol ., 202: 3295, 1999; Vollrath, F & Knight, DP, Nature 410: 541, 2001). Thus, dragline spider webs have received much attention as materials with various industrial applications. In addition, since spider webs are biocompatible and biodegradable, many applications in biomedical fields are expected. Unfortunately, since spiders have strong and aggressive area protection instincts, natural dragline spider webs cannot be easily obtained by spider culture or the like.
- the present inventors have co-expressed glycine tRNA as a result of diligently trying to provide a dragline recombinant silk protein having high molecular weight and similar physical properties to natural silk protein when manufactured from fibers, such as dragline silk protein produced from spider.
- a recombinant silk protein having a high molecular weight of 284.9kDa or more from bacteria such as Escherichia coli it was confirmed that it can be made of spider web fibers having better physical properties than the existing natural spider web fibers by spinning it, and completed the present invention. It was.
- Another object of the present invention is to provide cobweb fibers with improved physical properties spun with high molecular weight recombinant silk protein.
- the present invention provides a high molecular weight recombinant silk or silk-like protein having a structure in which a glycine content of 10% or more is repeated 64 to 160 times.
- the present invention also provides a high molecular weight recombinant silk protein having a structure in which the peptide of SEQ ID NO: 1 is repeated 64 to 160 times and having a molecular weight of 192.8 to 482 kDa.
- the present invention also provides a method for producing a high molecular weight recombinant silk or silk-like protein, characterized in that simultaneously expresses the gene encoding the recombinant silk or silk-like protein and the base sequence encoding the glycine tRNA.
- the present invention also provides a method for producing micro- or nano-sized cobweb or cobweb-like fibers, which comprises spinning a solution comprising the high molecular weight recombinant silk or silk-like protein.
- the present invention also provides micro- or nano-sized cobwebs or cobweb-like fibers, which are produced by the above method.
- the present invention also has a structure in which the peptide of SEQ ID NO: 1 is repeated 64 to 160 times, and the micro- or nano-sized spider web fibers, characterized in that spinning a solution containing a recombinant silk protein having a molecular weight of 192.8 to 482 kDa It provides a method of manufacturing.
- the present invention also provides micro- or nano-sized cobweb fibers, which are produced by the above method.
- Figure 1 shows the amino acid sequence of the expression structure and repeat unit for recombinant silk protein expression.
- FIG. 2 is a photograph showing the molecular weight of 16mer, 32mer, 64mer and 96mer recombinant silk protein separated from 10% SDS-PAGE gel.
- FIG. 3 is a graph showing stress-strain curves of fibers spun from 20% (w / v) recombinant silk protein solution using the wet spinning method.
- FIG. 4 is a graph showing fracture strain, tensile strength and Young's modulus of fibers spun from 20% (w / v) recombinant silk protein solution using a wet spinning method.
- 5 is a graph showing the tensile strength, strength and Young's modulus of the fiber according to the dope concentration of the recombinant silk protein.
- the term "silk protein” refers to a protein that is biosynthesized by a recombinant protein production method as a synthetic silk protein having a nearly similar molecular and structural profile to a natural silk protein, for example, dragline silk, silk fibroin (silk fibroin), flagelliform silk, etc. are mentioned.
- the term "silk-like protein” as used herein is a protein that is biosynthesized by a recombinant protein production method, including a peptide having a glycine content of 10% or more in a repeating unit, similar to silk protein, for example, elastin, secretary (byssus), collagen, etc. are mentioned.
- cobweb fibers is meant herein fibers that are almost similar to natural cobweb fibers, prepared using a synthetic silk protein synthesized, and "cobweb-like fibers” refers to cobweb fibers, prepared using a recombinant silk-like protein synthesized. It means a fiber having similar physical properties.
- a "recombinant protein” is inserted into a vector, ie, a self-replicating plasmid or virus, into the genomic DNA of a host cell, or in the form of a separate molecule in the host.
- a protein encoded by the nucleic acid sequence it means a protein in which the nucleic acid sequence is expressed.
- host cell means any cell capable of expressing a functional gene and / or gene product derived from another cell or organ.
- the present invention relates to a high-molecular weight recombinant silk or silk-like protein having a structure in which a peptide having a glycine content of 10% or more is repeated 64 to 160 times.
- the peptide having a glycine content of 10% or more constituting the silk protein or silk-like protein may be dragline silk, elastin, silk fibroin, byssus, flagella silk ( flagelliform silk) and collagen (collagen) may be characterized in that the repeating unit peptide constituting a protein selected from the group consisting of.
- the amino acid sequence of SEQ ID NO: 1 to 4 is a repeating unit peptide of dragline silk protein
- the amino acid sequence of SEQ ID NO: 5 to 7 is a repeating unit peptide of elastin
- the amino acid sequence of SEQ ID NO: 8 is a repeating unit peptide of silk fibroin
- the amino acid sequence of SEQ ID NO: 9 is a repeat unit peptide of the secretary
- the amino acid sequence of SEQ ID NO: 10 is a repeat unit peptide of flagella silk
- SEQ ID NOs: 11 and 12 are repeat unit peptides of collagen.
- the recombinant silk protein (hereinafter referred to as "64mer") prepared to include 64 times of the amino acid sequence represented by SEQ ID NO: 1 as a repeating unit has a molecular weight of about 192.8kDa, and has been synthesized in E. coli. It has been demonstrated that the production of recombinant silk proteins with molecular weights larger than the largest dragline silk protein (163 kDa) is possible.
- the recombinant silk protein (hereinafter referred to as "96mer") prepared to include 96 times with the amino acid sequence represented by SEQ ID NO: 1 as a repeat unit (hereinafter referred to as "96mer”), the molecular weight of 284.9kDa, obtained from spider It was confirmed to have a high molecular weight almost similar to the molecular weight (250 ⁇ 320kDa) of the natural silk protein.
- the recombinant silk or silk-like protein according to the present invention has a structure in which the peptide is repeated 64 to 160 times. It is characterized in that, preferably having a structure that is repeated 80 to 160 times, more preferably 96 to 160 times.
- amino acid sequence used as the repeating unit is not limited to the exact sequence of SEQ ID NOS: 1 to 12.
- amino acid sequence herein includes variants.
- amino acid sequences of the proteins of the invention also include all sequences that vary by insertion, deletion and substitution.
- amino acid “substitution” is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, that is to say conservative amino acid replacement.
- Amino acid substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the associated residues.
- nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine and polarly polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine
- positively charged (basic) amino acids include arginine, lysine, and histidine
- negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- “Insert” or “deleted” in the repeat unit typically ranges from about 1 to 5 amino acids, preferably about 1, 2 or 3 amino acids.
- the addition of amino acids increased due to the addition of amino acids inserted in the repeating unit is typically less than 100, preferably less than 80, more preferably less than 50, most preferably less than 20 amino acids, the present invention Is inserted into a repeating unit of and inserted into and / or added to the protein. It should be noted that the present invention contemplates only such additions that do not adversely affect the required properties of the proteins described herein.
- Acceptable modifications can be determined experimentally by using recombinant DNA techniques to systematically perform the insertion, deletion, or substitution of amino acids in a protein and to analyze the activity of the resulting recombinant variants. This does not require a person skilled in the art to go beyond routine experimentation.
- the present invention is characterized in that it comprises an amino acid sequence having at least 90% or more homology with SEQ ID NO: 1 as a repeating unit.
- SEQ ID NO: 1 as a repeating unit.
- homology refers to the degree of similarity between two amino acid sequences. Homologous means similar in sequence and functionality.
- Homology comparisons can be performed with the naked eye, but typically, an easily accessible sequence comparison program can be used to calculate percent homology between two or more sequences (Wilbur, WJ & Lipman, DJ, Proc. Natl. Acad. Sd). . USA, 80:. 726, 1983).
- the recombinant silk protein or recombinant silk-like protein may be prepared by co-expressing glycine tRNA in bacteria. Therefore, in another aspect, the present invention relates to a method for producing a recombinant silk or silk-like protein, which simultaneously expresses the gene encoding the recombinant silk protein or the recombinant silk-like protein and the base sequence encoding the glycine tRNA. .
- the bacterium may use E. coli. That is, unlike the production of high-molecular weight recombinant silk protein has not been reported previously, in the present invention, the base sequence encoding the glycine tRNA in bacteria such as E.
- the coli encodes a silk protein having the amino acid sequence of SEQ ID NO.
- the silk protein according to the present invention having the amino acid sequence of SEQ ID NO: 1 as a repeating unit has a molecular weight of 192.8 to 482 kDa.
- the present invention relates to a spider web or a spider web-like fiber having improved physical properties prepared by spinning the high molecular weight recombinant silk or silk-like protein.
- the dope solution containing the high-molecular weight recombinant silk protein or recombinant silk-like protein can be spun through the spinneret to produce micro- or nano-sized cobweb fibers.
- the dope solution is meant all liquid mixtures containing silk protein and are extruded for the formation or film casting of cobweb fibers.
- the dope solution may also contain higher order aggregates, such as, for example, dimers, trimers, and tetramers, in addition to the protein chain, considering this as a monomer.
- the dope solution is an aqueous solution of pH 4.0-12.0 and has less than 40% (w / v) organic or chaotropic agent.
- the dope solution does not contain any organic solvents or chaotropic agents, but may contain additives for improving the shelf life, stability or workability of the solution.
- the dope solution may be characterized in that it comprises 20 to 80% (w / v) of the recombinant silk protein.
- the dope solution may be characterized in that the liquid is spun into a liquid coagulation bath, preferably the liquid coagulation bath is methanol, ethanol, isopropanol, acetonitrile, water (water) and water-soluble ammonium sulfate ( It may be characterized by containing a liquid selected from the group consisting of aqueous ammonium sulfate).
- the diameter of the produced spider web fibers is determined by the diameter of the exit protrusion, the spider web fibers may be 0.6 ⁇ 50 ⁇ m in diameter, but is not limited thereto.
- spider web having improved physical properties by using a high molecular weight recombinant silk protein according to the present invention It was confirmed that the provision of fibers is possible.
- fibers made from 96mer recombinant silk protein showed a tensile strength of 508 ⁇ 108 MPa and a breakage strain of 15 ⁇ 5%, indicating that spider N. clavipes spider dragline silk (740-1,200 MPa; 18 -27%).
- the Young's modulus of the 96mer fibers was 21 ⁇ 4 GPa, which was twice the Young's modulus of natural dragline silk (11-14 GPa).
- the tensile strength (508 ⁇ 108 MPa) of 96mer fibers in the present invention is the highest tensile strength of the recombinant cobweb proteins reported to date.
- the fiber can be produced by an electrospinning method that sprays in the direction of an applied electric field by applying a voltage to a solution containing the high molecular weight recombinant silk protein or recombinant silk-like protein. Nano-sized cobweb or cobweb-like fibers can be obtained in this way.
- each of the recombinant silk proteins can be dissolved in hexafluoroisopropanol (HFIP; Sigma) at room temperature for two days to obtain a 12% (w / v) silk solution.
- HFIP hexafluoroisopropanol
- a drop of silk solution at the tip of a glass pipette with a platinum wire electrode is applied at a voltage of 12 kV, and when the applied electric force exceeds the surface tension of a drop of silk solution, a fiber jet is formed and sprayed in the direction of the applied field. do.
- the fibers can be collected on a glass material on a flat plate covered with aluminum, and some electrospun fibers can be treated with methanol to cause beta-side formation.
- the air can be dried for 3 days and then the physical properties can be measured.
- the elastic modulus of each sample used can be calculated.
- the force was measured using a silicon cantilever (FESP, Veeco Instruments Inc.) using an atomic force microscope, Dimension V (Veeco Instruments Inc., Plainview, NY). The distance curve is drawn. Approximately 20 measurements are made for each specimen.
- an extended Hertz model can be applied to each force curve (Hertz, 1882, Sneddon, 1965). The elasticity is calculated directly from the derived application variables. In the Hertz model, the modulus of elasticity E is given by
- F, v, a, k, and d indicate the force at the tip, the ratio of poison, the deformation of the sample, the spring constant of the cantilever, and the deflection of the cantilever.
- the elastic modulus of the fiber can be obtained from information such as the shape of the tip, the spring constant, the type and deflection of the cantilever tip, and the poison ratio of the specimen.
- the recombinant silk protein / recombinant silk-like protein as defined herein and the fibers, filaments, films, foams, spheres, nanofibrils, hydrogels, etc. prepared therefrom are in the fields of biotechnology and / or medicine, preferably It may be used for the manufacture of wound sutures or coating systems, for the production of suture materials for use in neurosurgery or ophthalmic surgery.
- protein / seal may be used for the production of alternative materials, preferably artificial cartilage or tendon materials.
- cobweb fibers or cobweb-like fibers of the present invention may be used for medical adhesive garment fabrics, such as medical adhesive strips, skin grafts, replacement ligaments, surgical meshes, vest linings, container fabrics, bags or purses, Wide range of industrial and industrial products, including cables, ropes, adhesive bonding materials, non-adhesive bonding materials, leash materials, car covers and parts, aircraft construction materials, weathering materials, fluid partition materials, sports equipment
- the manufacturing and virtually required properties of the can be used for the use of almost any fiber or fabric, which is of high tensile strength and elasticity.
- the adaptability and use of fiber products that are stable in other forms, such as dry spray coatings, bead like particles, or the like, or use in mixtures with other compositions, is also contemplated herein.
- the recombinant silk protein and recombinant silk like protein of the present invention can be added to cellulose and keratin and collagen products, so the present invention also provides paper or skin comprising cellulose and / or keratin and / or collagen and the recombinant protein of the invention.
- the present invention also provides paper or skin comprising cellulose and / or keratin and / or collagen and the recombinant protein of the invention.
- Paper and skin care and hair care products, into which the proteins of the invention have been introduced exhibit improved properties, in particular improved tensile strength or tear strength.
- the high molecular weight recombinant protein of the present invention can be used as a coating for textiles and leather articles, thereby imparting stability and durability of the coated articles.
- Silk proteins show particular applicability to leather article coatings, in which case they can avoid or at least reduce tanning and adverse effects on the environment.
- Example 1 Expression Vectors of High Molecular Weight Recombinant Silk Proteins Construction of Expression Vectors of pSH32, pSH48, pSH64, pSH80, and pSH96 and Glycine TRNAs and Preparation of Recombinant Silk Proteins of Various Molecular Weights
- a 1.7 kb fragment obtained by cleaving plasmid pSH16a with restriction enzymes Spe I and Nhe I was linked to plasmid pSH32 cut with restriction enzyme Spe I, thereby recombining plasmid pSH48 with plasmid pSH32 with restriction enzymes Sp eI and Nhe I.
- the cut 3.4 kb fragment was ligated with plasmid pSH32 cut with restriction enzyme Spe I to obtain a recombinant plasmid pSH64 comprising a nucleic acid sequence encoding the 64mer silk protein of SEQ ID NO: 15. Each inserted direction was confirmed by cutting with restriction enzymes Spe I and Nhe I.
- E. coli W3110 (derived from E. coli K- 12, ⁇ -, F -, prototrophic) using a separate chromosome from the strain as a template and SEQ ID NO: 19 and SEQ ID NO: 20 PCR was performed using primers.
- PCR was performed using Pfu polymerase (SolGent, Korea), and the reaction conditions were as follows. The first denaturation was performed once at 95 ° C for 4 minutes, after which the second denaturation was carried out at 95 ° C for 20 seconds, the annealing at 51 ° C for 30 seconds, and the extension at 72 ° C for 60 seconds. This was repeated 10 times. And further 19 times was repeated denaturation for 20 seconds at 95 °C, 30 seconds binding at 60 °C, 60 seconds extension at 72 °C. This was followed by one last extension at 72 ° C. for 5 minutes.
- DNA obtained by the above PCR was subjected to agarose gel electrophoresis to obtain a purified 479 bp PCR product.
- agarose gel electrophoresis To cut it simultaneously with restriction enzymes Bam HI and Eco RV (New England Biolabs, USA), and to use a promoter of a tetracycline resistance gene ( tet ) that can be expressed continuously, plasmid pACYC184 (New England Biolabs, USA) ) Were also digested with the same restriction enzymes.
- the cleaved PCR product and the plasmid were ligation with a T4 DNA ligase (Roche, Germany), which was then transformed into Escherichia coli Top10 (F - mcr A ⁇ ( mrr - hsd RMS- mcr BC) ⁇ 0 lac Z ⁇ M15 ⁇ lac X74 rec A1 ara D139 ⁇ (ara - leu) was transformed to 7697 gal U gal K rps L ( Str R) end A1 nup G).
- the transforming strain was selected from LB agar solid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, agar 15 g / L) containing 34 mg / L chloramphenicol. Plasmid pTet-glyVXY was obtained. The produced recombinant plasmid was cut and confirmed by restriction enzyme and confirmed by sequencing.
- PCR was performed using Pfu polymerase (SolGent, Korea), and the reaction conditions were as follows. The first denaturation was performed once at 95 ° C for 3 minutes, after which the second denaturation was carried out at 95 ° C for 20 seconds, the annealing at 52 ° C for 30 seconds, and the extension at 72 ° C for 50 seconds. This was repeated 10 times. And further 19 times was repeated denaturation for 20 seconds at 95 °C, binding for 30 seconds at 62 °C, 50 seconds extension at 72 °C. This was followed by one last extension at 72 ° C. for 5 minutes.
- DNA obtained by the PCR was subjected to agarose gel electrophoresis to obtain a purified 674 bp PCR product.
- the 647 bp PCR product and plasmid pTet-glyVXY were digested with restriction enzymes Sph I and Sal I (New England Biolabs, USA), respectively, and linked with T4 DNA ligase (Roche, Germany). Transformed.
- the transforming strain was selected from LB agar solid medium (tryptone 10 g / L, yest extract 5 g / L, NaCl 5 g / L, agar 15 g / L) containing 34 mg / L chloramphenicol. Plasmid pTet-gly2 was obtained. The produced recombinant plasmid was cut and confirmed by restriction enzyme and confirmed by sequencing.
- the pSH16a vector and pSH32 vector prepared in Example 1-1 were prepared using pTet-glyVXY and E. coli BL21 (DE3) ( F - omp T hsd SB obtained in Example 1-2, respectively).
- (rB - mB -) was charged to a gal dcm (DE3) a prophage carrying the T7 RNA polymerase gene) (New England Biolabs, USA).
- the pSH64 vector and pSH96 vector were put into the pTetgly2 vector and E. coli BL21 (DE3) obtained in Example 1-3, respectively.
- the transformed strains were LB liquid medium containing 34 mg / L chloramphenicol and 25 mg / L kanamycin (tryptone 10 g / L, yest extract 5 g / L, NaCl 5 g / L). Inoculated to and incubated continuously shaking at 180 rpm at 30 °C.
- Silk protein gene expression was induced by adding 1 mM IPTG when the optical density (OD) measured at 600 nm wavelength by spectrophotometer after 1% inoculation was 0.2, 0.4 or 0.6. Cultures were harvested 5 hours after induction expression.
- the collected culture solution was centrifuged at 10,000 g for 10 minutes at 4 ° C to obtain a cell pellet, which was dissolved in TE buffer and 5x Laemmli sample buffer. Equal amounts (0.024 mg) of sample were separated using 10% SDS-PAGE, stained with Coomassie brilliant blue R250 (Bio-Rad, USA) solution, using GS-710 Calibrated Imaging Densitometer (Bio-Rad, USA) The total protein amount was determined according to Bradford's protein quantification method, and the amount of the protein in the solution was determined by calculating Bovine serum albumin as the reference solution (Bradford, MM, Anal. Biochem., 72: 248, 1976).
- the recombinant silk proteins of each of 16mer (using pSH16a vector), 32mer (using pSH32 vector), 64mer (using pSH64 vector) and 96mer (using pSH96 vector) were about 50.4, 100.7, and 192.8, respectively.
- a molecular weight of 284.9 kDa that is, the largest drag line silk proteins but its molecular weight is known to be high than this was only a 163kDa difficult the production of a silk protein having a molecular weight of the synthesized in E. coli and to date (Fahnestock, SR & Irwin, SL, Appl. Microbiol.
- the dope solution was pumped at a rate of 1 to 2 ml per hour. Injection molding was performed using (KDS100; KD Scientific). The concentration of the dope solution was 20% (w / v) of the maximum working concentration of the naturally sized 96mer protein due to solubility and elasticity, and all of the silk protein was spun at a concentration of 20% (w / v).
- the fibers were then dried at room temperature, and the tension that the fibers were subjected to was sustained because they needed to prevent shrinkage and maintain extended lengths for measurement.
- Statistical tests were performed using the Unpaired t-test and considered statistically significant when P ⁇ 0.05.
- spider webs made of 96mer recombinant silk protein of 284.9kDa it is unexpected in all areas of fracture strain, tensile strength and Young's modulus in view of the increase in spider web made of recombinant silk protein of 64mer or less. It was found to increase significantly.
- the fiber made from 96mer recombinant silk protein showed tensile strength of 508 ⁇ 108 MPa and fracture strain of 15 ⁇ 5%, which is natural spider dragline silk (740-1,200 MPa; 18 of N. clavipes) . -27%).
- the Young's modulus of 96mer fibers was 21 ⁇ 4 GPa, which was twice the Young's modulus of natural dragline silk (11-14 GPa).
- the tensile strength (508 ⁇ 108 MPa) of 96mer fibers in the present invention is the highest tensile strength of the recombinant cobweb proteins reported to date.
- the present invention has the effect of providing a high molecular weight recombinant silk or silk-like protein having a molecular weight almost similar to that of natural silk protein and micro or nano-sized spider web or spider web-like fiber having improved physical properties.
- Recombinant silk protein and recombinant silk-like protein according to the present invention has a high molecular weight, such as dragline silk protein produced from the spider has the advantage of having similar or better physical properties than the natural silk protein when manufactured from fibers. Therefore, the recombinant silk protein and the recombinant silk pseudoprotein according to the present invention, and the fibers prepared using the same, are very useful because they can be used in various industrial fields such as biotechnological and / or pharmaceutical fields where application of spider web fibers is expected. .
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Description
Claims (17)
- 글리신 함량이 10% 이상인 펩타이드가 64~160회 반복되어 있는 구조를 가지는 고분자량의 재조합 실크 또는 실크 유사 단백질.
- 제1항에 있어서, 상기 펩타이드가 80~160회 반복되어 있는 구조를 가지는 고분자량의 재조합 실크 또는 실크 유사 단백질.
- 제1항에 있어서, 상기 펩타이드가 96~160회 반복되어 있는 구조를 가지는 고분자량의 재조합 실크 또는 실크 유사 단백질.
- 제1항에 있어서, 상기 펩타이드는 드레그라인 실크 (dragline silk), 엘라스틴(elastin), 실크 피브로인(silk fibroin), 비서스(byssus), 편모상 실크(flagelliform silk) 및 콜라겐(collagen)로 구성된 군에서 선택된 단백질을 구성하는 반복단위 펩타이드인 것을 특징으로 하는 고분자량의 재조합 실크 또는 실크 유사 단백질.
- 제1항에 있어서, 상기 펩타이드는 서열번호 1 내지 11 중 어느 하나의 아미노산 서열을 가지는 것을 특징으로 하는 고분자량의 재조합 실크 또는 실크 유사 단백질.
- 제5항에 있어서, 상기 펩타이드와 적어도 90% 이상의 상동성을 가지는 아미노산 서열이 반복되어 있는 것을 특징으로 하는 고분자량의 재조합 실크 또는 실크 유사 단백질.
- 서열번호 1의 펩타이드가 64~160회 반복되어 있는 구조를 가지고, 분자량이 192.8 내지 482kDa인 재조합 실크 단백질.
- 제1항 내지 제7항 중 어느 한 항의 재조합 실크 또는 실크 유사 단백질을 코딩하는 유전자와 글리신 tRNA를 코딩하는 염기서열을 박테리아에서 동시에 발현시키는 것을 특징으로 하는 고분자량의 재조합 실크 또는 실크 유사 단백질의 제조방법.
- 제8항에 있어서, 상기 박테리아는 대장균인 것을 특징으로 하는 재조합 실크 또는 실크 유사 단백질의 제조방법.
- 제1항 내지 제6항 중 어느 한 항의 고분자량의 재조합 실크 또는 실크 유사 단백질을 포함하는 도프 용액을 방사하는 것을 특징으로 하는 마이크로 크기 또는 나노 크기의 거미줄 또는 거미줄 유사 섬유를 제조하는 방법.
- 제10항에 있어서, 20~80%(w/v)의 재조합 실크 또는 실크 유사 단백질을 포함하는 도프 용액을 습식방사하는 것을 특징으로 하는 방법.
- 제11항에 있어서, 상기 도프 용액은 액체응고조로 방적되는 것을 특징으로 하는 방법.
- 제12항에 있어서, 상기 액체응고조는 메탄올, 에탄올, 이소프로판올, 아세토니트릴(acetonitrile), 물(water) 및 수용성 황산암모늄(aqueous ammonium sulfate)로 구성된 군에서 선택되는 액체를 함유하는 것을 특징으로 하는 방법.
- 제10항의 방법에 의하여 제조되는 것을 특징으로 하는 마이크로 크기 또는 나노 크기의 거미줄 또는 거미줄 유사 섬유.
- 제7항의 고분자량의 재조합 실크 단백질을 포함하는 도프 용액을 방사하는 것을 특징으로 하는 마이크로 크기 또는 나노 크기의 거미줄 섬유를 제조하는 방법.
- 제15항의 방법에 의하여 제조되는 것을 특징으로 하는 마이크로 크기 또는 나노 크기의 거미줄 섬유.
- 제16항에 있어서, 적어도 252MPa의 장력 및 10.14GPa이상의 영계수 및 4.31%이상의 파괴변형도를 가지는 것을 특징으로 하는 거미줄 섬유.
Priority Applications (4)
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US13/124,818 US20120231499A1 (en) | 2010-03-11 | 2011-03-11 | High-molecular-weight recombinant silk or silk-like protein and micro- or nano-sized spider silk or silk-like fiber produced therefrom |
CN201180017801.3A CN102906107B (zh) | 2010-03-11 | 2011-03-11 | 高分子量重组丝或类丝蛋白、以及使用它们制造的微米或纳米级蜘蛛丝或者类蜘蛛丝纤维 |
EP11713459.3A EP2546263A4 (en) | 2010-03-11 | 2011-03-11 | RECOMBINANT SILK MUG OR SILKY PROTEIN OF HIGH MOLECULAR WEIGHT, AND MICRO OR NANOSCALE SPIDER FIBER OR SPINNING FIBER PRODUCED FROM THE RECOMBINANT SILK PROTEIN OR SILKY PROTEIN |
JP2012556990A JP5858936B2 (ja) | 2010-03-11 | 2011-03-11 | 高分子量の組み換えシルク蛋白質、またはシルク様蛋白質、及びこれを利用して製造されたマイクロ、またはナノサイズのクモの巣線維、またはクモの巣様繊維 |
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KR1020100021934A KR101317420B1 (ko) | 2010-03-11 | 2010-03-11 | 고분자량의 재조합 실크 또는 실크 유사 단백질 및 이를 이용하여 제조된 마이크로 또는 나노 크기의 거미줄 또는 거미줄 유사 섬유 |
KR10-2010-0021934 | 2010-03-11 |
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US (1) | US20120231499A1 (ko) |
EP (1) | EP2546263A4 (ko) |
JP (1) | JP5858936B2 (ko) |
KR (1) | KR101317420B1 (ko) |
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WO (1) | WO2011112046A2 (ko) |
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Cited By (4)
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CN103833838A (zh) * | 2012-11-22 | 2014-06-04 | 中国科学院青岛生物能源与过程研究所 | 一种高性能类蛛丝蛋白材料及其生物合成方法 |
CN103833838B (zh) * | 2012-11-22 | 2016-06-29 | 中国科学院青岛生物能源与过程研究所 | 一种高性能类蛛丝蛋白材料及其生物合成方法 |
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Publication number | Publication date |
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WO2011112046A3 (ko) | 2012-01-05 |
CN102906107B (zh) | 2015-05-20 |
KR20110102757A (ko) | 2011-09-19 |
KR101317420B1 (ko) | 2013-10-10 |
US20120231499A1 (en) | 2012-09-13 |
EP2546263A4 (en) | 2013-07-31 |
JP2013528568A (ja) | 2013-07-11 |
EP2546263A2 (en) | 2013-01-16 |
CN102906107A (zh) | 2013-01-30 |
JP5858936B2 (ja) | 2016-02-10 |
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