JP5883271B2 - Method for producing artificial polypeptide ultrafine fiber - Google Patents

Description

The present invention relates to a process for the production of artificial polypeptide microfine textiles, which is a kind of synthetic protein fibers.

  The spider fiber is a fiber having high strength and toughness, and is known to have higher strength and toughness than high-strength steel, nylon 6 fiber, aramid fiber, carbon fiber and the like. In addition, there is an advantage that the raw material can be converted to biomass without using petroleum. Some artificial spider silk fibers have also been proposed. For example, Patent Document 1 proposes that a synthetic protein spinning solution is wet-spun and extruded into a coagulation bath composed of 90% methanol to obtain fibers. Patent Document 2 proposes the present fiber which is wet-spun and has a tensile strength of 200 MPa or more. Non-Patent Document 1 discloses a drawn yarn having a strength of 1.91 to 2.26 g / d and an elongation of 43.4 to 59.6% by wet spinning and a draw ratio of 5 in a methanol bath and a water bath. . Non-Patent Document 2 discloses a drawn yarn having a stress of 600 MPa and an elongation of 25% by wet spinning and setting the draw ratio to 5 times. Non-Patent Document 3 discloses a drawn yarn having a stress of 280 to 350 MPa and an elongation of 30 to 40% by retaining a wet-spun undrawn yarn in steam for 5 minutes and drawing it five times.

  However, conventional artificial spider fibers are manufactured by wet spinning, and it is difficult to obtain ultrafine fibers by the wet spinning method. Particularly, fibers having a diameter of 8 μm (single fiber fineness of about 1 deci tex) or less are stably used. It was difficult to get.

JP-T-2004-503204 Special table 2009-521921

Anthoula Lazaris, et.al., "Spider Silk Fiber Spun from Soluble Recombinant Silk Produced in Mammalian Cells", Science, 295, p472, January 18, 2002 Xiao-Xia Xia, et.al., "Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coil results in a strong fiber", PNAS, vol. 107, No. 32, p14059-14063, August 10, 2010 Day M. Elice, et.al., "Bioinspired Fibers Follow the Track of Natural Spider Silk", Macromolecules, Vol. 44, No. 5, p1166-1176, April 2, 2011

The present invention for solving the above conventional problems, to provide a method for manufacturing a thin artificial polypeptide microfine textiles of single fiber diameters.

Method for producing artificial polypeptide microfibrous sheet of the invention, a method for making a human-forming polypeptide microfibrous sheet a spinning solution that is obtained by electrospinning comprising a polypeptide derived from nature spider silk protein (spider silk proteins) A step of dissolving the polypeptide in a solvent to form a spinning solution ; a step of forming the fiber by discharging the spinning solution from a base to which voltage is applied by electrospinning; The average diameter of single fibers of the ultrafine fibers is 1 μm or less, and the single fiber diameter of the ultrafine fibers varies between 0.1 and 1 μm, and the ultrafine fibers themselves It is characterized by making it a sheet | seat by entanglement of .

  The present invention is to form a fiber by discharging a spinning solution in which a polypeptide derived from a natural spider silk protein is dissolved in a solvent by electrospinning from a base to which voltage is applied, and by detaching the solvent from the fiber, Ultrafine fibers that cannot be obtained by wet spinning can be obtained.

FIG. 1 is an explanatory view of an electrospinning apparatus according to an embodiment of the present invention. FIG. 2 is an explanatory view of an electrospinning apparatus according to another embodiment of the present invention. FIG. 3 is a stress-displacement (strain) curve of the artificial polypeptide ultrafine fiber sheet obtained in Example 1 of the present invention. FIG. 4 is a stress-displacement (strain) curve of the artificial polypeptide ultrafine fiber sheet obtained in Example 2 of the present invention. FIG. 5 is a scanning electron microscope (SEM) photograph of the artificial polypeptide ultrafine fiber sheet obtained in Example 2 of the present invention.

(1) Polypeptide In the present invention, a polypeptide derived from a natural spider silk protein is used as a raw material. The polypeptide is not particularly limited as long as it is derived from a natural spider silk protein. From the viewpoint of excellent toughness, the polypeptide is preferably a polypeptide derived from a large sputum bookmark thread protein produced in a spider large bottle-like line. Examples of the large sputum bookmark thread protein include large bottle-shaped spidroin MaSp1 and MaSp2 derived from the American spider spider (Nephila clavipes), ADF3 and ADF4 derived from the Araneus diadematus, and the like. The polypeptide derived from the large sputum bookmark thread protein includes a mutant, analog or derivative of the large sputum bookmark thread protein.

  Examples of the polypeptide derived from the large sputum dragline protein include a polypeptide comprising 2 or more, preferably 5 or more, more preferably 10 or more amino acid sequence units represented by Formula 1: REP1-REP2 (1). Can be mentioned. Alternatively, the polypeptide derived from the large sputum dragline protein includes an amino acid sequence unit represented by Formula 1: REP1-REP2 (1), and the C-terminal sequence is any one of SEQ ID NOs: 1 to 3. The polypeptide may be an amino acid sequence shown or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3. In addition, in the polypeptide derived from the large sputum dragline protein, the units of the amino acid sequence represented by the formula (1): REP1-REP2 (1) may be the same or different. From the viewpoint of productivity, the polypeptide derived from the large sputum bookmark thread protein preferably has a molecular weight of 500 kDa or less, more preferably 300 kDa, from the viewpoint of productivity when producing a recombinant protein using a microorganism such as Escherichia coli as a host. Or less, more preferably 200 kDa or less.

  In the formula (1), REP1 means polyalanine, and alanine is continuously 2 to 20 residues, more preferably 3 to 16 residues, still more preferably 4 to 12 residues, most preferably It is an amino acid sequence in which 5 to 8 residues are arranged. In the formula (1), REP2 is an amino acid sequence consisting of 10 to 200 amino acids, and the total number of residues of glycine, serine, glutamine and alanine contained in the amino acid sequence is the total number of amino acid residues. On the other hand, it is 40% or more, preferably 60% or more, more preferably 70% or more.

  In the large discharge tube bookmark yarn, the REP1 corresponds to a crystal region forming a crystal β sheet in the fiber, and the REP2 is an amorphous type which is more flexible in the fiber and largely lacks a regular structure. Corresponds to the area. [REP1-REP2] corresponds to a repetitive region (repetitive sequence) composed of a crystal region and an amorphous region, and is a characteristic sequence of a bookmark thread protein.

  The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287), and the amino acid sequence shown in SEQ ID NO: 2 is The amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus, and the amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1. Is the same.

  Formula 1: REP1-REP2 (1) is included, and the C-terminal sequence is represented by any one of SEQ ID NOS: 1-3 or SEQ ID NO: 1-3 As a polypeptide consisting of an amino acid sequence having 90% or more homology with the amino acid sequence, for example, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 can be used. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is an amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, His10 tag and HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminus. The first to thirteenth repetitive regions are increased so as to be approximately doubled, and the translation is mutated to terminate at the 1154th amino acid residue. In the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, the C-terminal sequence is identical to the amino acid sequence shown in SEQ ID NO: 3.

  In addition, the amino acid sequence comprising a unit of the amino acid sequence represented by the formula 1: REP1-REP2 (1) and the C-terminal sequence is represented by any one of SEQ ID NOs: 1 to 3, or any one of SEQ ID NOs: 1 to 3 As a polypeptide comprising an amino acid sequence having 90% or more homology with the amino acid sequence shown, one or more amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, inserted and / or added. A protein having an amino acid sequence and a repeating region consisting of a crystalline region and an amorphous region can be used. In the present invention, “one or more” means, for example, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or Mean one or several. In the present invention, “one or several” means 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, Means two or one.

  A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 (hereinafter referred to as ADF3kai-F) comprises an initiation codon, His10 tag and HRV3C protease recognition site at the N-terminus of the amino acid sequence of ADF3 (GI: 1263287). In the amino acid sequence (SEQ ID NO: 7) in which the amino acid sequence (SEQ ID NO: 5) has been added and 29 amino acid residues have been deleted from the C terminus, ie, the amino acid sequence shown in SEQ ID NO: 10 has been placed at the C terminus. The 22nd, 31st and 32nd Gln are replaced with Lys, and the 26th arginine (Arg) is replaced with Lys.

  The polypeptide can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for producing the gene is not particularly limited, and a gene encoding a natural spider silk protein is amplified and cloned from a spider-derived cell by polymerase chain reaction (PCR) or the like, or chemically synthesized. The method for chemically synthesizing the gene is not particularly limited. For example, based on the amino acid sequence information of the natural spider silk protein obtained from the NCBI web database, etc., AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) Oligonucleotides automatically synthesized by a company) can be synthesized by ligation by PCR or the like. At this time, in order to facilitate the purification and confirmation of the protein, a gene encoding a protein consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N terminus of the above amino acid sequence may be synthesized. . As the expression vector, a plasmid, fuzzy, virus or the like capable of expressing a protein from a DNA sequence can be used. The plasmid type expression vector is not particularly limited as long as it can express the target gene in the host cell and can be amplified by itself. For example, when Escherichia coli Rosetta (DE3) is used as a host, a pET22b (+) plasmid vector, a pCold plasmid vector, or the like can be used. Among these, it is preferable to use a pET22b (+) plasmid vector from the viewpoint of protein productivity. As the host, for example, animal cells, plant cells, microorganisms and the like can be used.

(2) Spinning solution The spinning solution (dope solution) is prepared by adding a solvent to the polypeptide and adjusting the viscosity to allow spinning. For example, when the polypeptide is derived from a Dwarf Spider, any solvent can be used as long as it can dissolve the polypeptide. Examples include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), formic acid, urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride, lithium thiocyanate, etc. In addition, the solution viscosity is 100 to 10,000 cP (centipoise). This is the spinning solution. Examples of the alkali metal halide include LiCl and NaCl.

(3) Electrospinning The electrospinning method (electrostatic spinning method) applies a voltage between a supply-side electrode (which can also be used as a spinneret) and a collection-side electrode (for example, a metal roll or a metal net). An electric charge is applied to the extruded dope solution and blown off to the collection side electrode. At this time, the dope solution is stretched to form fibers. In the present invention, a preferable applied voltage is 5 to 100 kV, more preferably 10 to 50 kV. When the voltage is less than 5 kV, there is resistance between the electrodes in the space (between the electrodes) in the atmosphere, so that the flow of electrons becomes poor and the dope solution is difficult to be charged. On the other hand, if it exceeds 100 kV, sparking occurs between the electrodes, and the dope solution may be ignited. The distance between the electrodes is preferably 1 to 25 cm, more preferably 2 to 20 cm. If the distance between the electrodes is less than 1 cm, sparks are likely to occur between the electrodes, and the dope solution may be ignited. If it exceeds 25 cm, the resistance between the electrodes is increased, the flow of electrons is hindered, It becomes difficult to be charged.

  FIG. 1 is an explanatory diagram of an electrospinning apparatus 10 according to an embodiment of the present invention. First, a voltage is applied by a power source 5 between the metal base nozzle 3 and the metal roll 4. The dope solution 2 is placed in the microsyringe 1 and moved in the direction of arrow P using a syringe pump, the dope solution 2 is pushed out from the metal nozzle 3, and the dope solution is blown off by electric charge to form a fibrous material 6. It rolls up simultaneously with depositing on the surface of the roll 4. Reference numeral 7 denotes a wound sheet-like (nonwoven fabric) ultrafine fiber. The sheet-like (nonwoven fabric) ultrafine fibers then desorb the solvent. The method for desorbing the solvent is preferably drying under reduced pressure or immersion in a solvent removal tank.

  FIG. 2 is an explanatory diagram of an electrospinning apparatus 20 according to another embodiment of the present invention. The difference from FIG. 1 is that a metal net 8 is used as a collection-side electrode. A voltage is applied between the spinneret nozzle 3 of the supply side electrode and the metal net 8, and the dope solution is stretched by electric charges to form a fibrous material 6, which is deposited on the surface of the metal net 8. Reference numeral 9 denotes a deposited sheet-like (non-woven fabric) ultrafine fiber. The sheet-like (nonwoven fabric) ultrafine fibers then desorb the solvent. The method for desorbing the solvent is preferably drying under reduced pressure or immersion in a solvent removal tank. The advantage of using a metal net is that metal nets of various shapes can be used. For example, a sheet-like (non-woven fabric) ultrafine fiber can be formed along the shape of the net itself, such as a bowl shape, a ball shape, and a wave shape. Since the fibers of the present invention are extremely fine, the fibers themselves are intertwined to form a sheet.

  The artificial polypeptide fiber of the present invention has an average diameter of 1 μm or less, preferably in the range of 0.1 to 1 μm. If it is the said range, a fiber can be obtained stably. A more preferable average diameter is in the range of 0.2 to 0.9 μm, and further preferably in the range of 0.3 to 0.8 μm. Moreover, the single fiber diameter of the ultrafine fiber of this invention is fluctuate | varied between 0.1-1 micrometer. This is a phenomenon unique to electrospinning. The specific gravity of the fiber of the present invention is 1.34. When calculating the fineness (unit: tex or deci tex), when the fiber cross section is round, it is calculated from the cross-sectional area, specific gravity, and length calculated from the fiber diameter. The artificial polypeptide fiber of the present invention is not limited to a circular cross section, and includes various shapes, and thus means an average diameter when the cross section is assumed to be circular. The fiber diameter varies within the above range.

  The polypeptide is preferably a polypeptide derived from ADF3, which is one of the two main dragline proteins of Araneus diadematus. This polypeptide is also advantageous in that it is basically high in elongation and toughness and easy to synthesize.

The artificial polypeptide fiber of the present invention may be chemically cross-linked between the polypeptide molecules in the fibroin fiber after drawing. Examples of functional groups that can be used for cross-linking in polypeptides include amino groups, carboxyl groups, thiol groups, and hydroxy groups, but are not limited thereto. The amino group of the lysine side chain contained in the polypeptide can be cross-linked with an amide bond by dehydration condensation with the carboxyl group of the glutamic acid or aspartic acid side chain. Crosslinking may be carried out by dehydration condensation reaction under vacuum heating, or by dehydration condensation agent such as carbodiimide. Moreover, you may use crosslinking agents, such as glutaraldehyde. Moreover, it can also bridge | crosslink with enzymes, such as transglutaminase. As an example, the crosslinking reaction may be performed with a crosslinking agent such as carbodiimide and glutaraldehyde. The carbodiimide is represented by the general formula R ¹ N═C═NR ² (where R ¹ and R ² represent an organic group containing an alkyl group having 1 to 6 carbon atoms or a cycloalkyl group), and the specific compound is 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N'-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide, diisopropylcarbodiimide (DIC), etc. There is. Among these, EDC and DIC are preferable because they have a high amide bond forming ability of peptide chains and easily undergo a crosslinking reaction. The crosslinking treatment is preferably performed by applying a crosslinking agent to the drawn yarn and polymerizing by vacuum heat drying. A 100% product of the crosslinking agent may be imparted to the fiber, or it may be diluted with a lower alcohol having 1 to 5 carbon atoms or a buffer solution and imparted to the fiber at a concentration of 0.005 to 10% by mass. The treatment conditions are preferably a temperature of 20 to 45 ° C. and a time of 3 to 42 hours. By the crosslinking treatment with the crosslinking agent, the strength, toughness, chemical resistance, etc. of the artificial polypeptide fiber can be increased.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited to the following Example.

Example 1
<Gene synthesis>
(1) Synthesis of ADF3Kai gene A partial amino acid sequence of ADF3 (GI: 1263287), one of the two major worm-coiled silkworm spider silk proteins, was obtained from the NCBI web database and started at the N-terminus of the same sequence. A gene encoding a protein consisting of an amino acid sequence (SEQ ID NO: 6) to which an amino acid sequence (SEQ ID NO: 5) consisting of a codon, a His10 tag and an HRV3C protease recognition site was added was commissioned to GenScript. As a result, a pUC57 vector (with an Nde I site immediately upstream of the 5 ′ end and an Xba I site immediately downstream of the 5 ′ end) into which the ADF3Kai gene having the base sequence shown in SEQ ID NO: 11 was introduced was obtained. Thereafter, the gene was treated with restriction enzymes with Nde I and EcoR I and recombined into a pET22b (+) expression vector.

(2) Synthesis of ADF3Kai-NRSH1 ADF3Kai amino acid sequence by site-directed mutagenesis using PrimeStar Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) using pET22b (+) expression vector introduced with ADF3Kai gene as a template. The codon GGC corresponding to the 632nd amino acid residue Gly of (SEQ ID NO: 6) was mutated to the stop codon TAA, and the ADF3Kai-NRSH1 gene shown in SEQ ID NO: 12 was constructed on pET22b (+). The accuracy of the introduction of the mutation was confirmed by a sequencing reaction using 3130xl Genetic Analyzer (Applied Biosystems). The amino acid sequence of ADF3Kai-NRSH1 is as shown in SEQ ID NO: 7.

(3) Synthesis of ADF3Kai-B ADF3Kai-B was synthesized by site-directed mutagenesis using PrimeStar Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) using the pET22b (+) expression vector introduced with the ADF3Kai-NRSH1 gene as a template. The codon CAA corresponding to the 22nd amino acid residue Gln of the amino acid sequence of NRSH1 (SEQ ID NO: 7) is mutated to the codon AAA encoding Lys, and the ADF3Kai-B gene shown in SEQ ID NO: 13 is transferred to pET22b (+). Built in. The accuracy of the introduction of the mutation was confirmed by a sequencing reaction using 3130xl Genetic Analyzer (Applied Biosystems). The amino acid sequence of ADF3Kai-B is as shown in SEQ ID NO: 8.

(4) Synthesis of ADF3Kai-F By site-directed mutagenesis using PrimeStar Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) using the pET22b (+) expression vector introduced with the ADF3Kai-B gene as a template, ADF3Kai- The codon CGA corresponding to the 26th amino acid residue Arg of the amino acid sequence of B (SEQ ID NO: 8) encodes the codon AAA encoding Lys, and the codon CAA corresponding to the 31st amino acid residue Gln encodes Lys. In codon AAA, codon CAA corresponding to the 32nd amino acid residue Gln was mutated to codon AAA encoding Lys, and the ADF3Kai-F gene shown in SEQ ID NO: 14 was constructed on pET22b (+). The accuracy of the introduction of the mutation was confirmed by a sequencing reaction using 3130xl Genetic Analyzer (Applied Biosystems). The amino acid sequence of ADF3Kai-F is as shown in SEQ ID NO: 9.

<Protein expression>
A pET22b (+) expression vector containing the ADF3Kai, ADF3Kai-NRSH1, ADF3Kai-B or ADF3Kai-F gene was transformed into E. coli Rosetta (DE3). After culturing the obtained single colony in 2 mL of LB medium containing ampicillin for 15 hours, 1.4 mL of the same culture solution was added to 140 mL of LB medium containing ampicillin and cultured at 37 ° C. and 200 rpm. The culture was continued until the OD ₆₀₀ was 3.5. Then, OD _{600 of} the culture broth of 3.5, added with 50% glucose 140mL in 2 × YT medium 7L containing ampicillin, and cultured until an OD ₆₀₀ of 4.0. Thereafter, protein expression was induced by adding isopropyl-β-thiogalactoviranoside (IPTG) to the obtained culture solution having an OD ₆₀₀ of 4.0 to a final concentration of 0.5 mM. At the time when 2 hours had elapsed since the addition of IPTG, the culture solution was collected by centrifugation. The protein solution prepared from the culture solution before and after the addition of IPTG was electrophoresed on a polyacrylamide gel, and depending on the addition of IPTG, the target sizes (ADF3Kai about 58.8 kDa, ADF3Kai-NRSH1 about 55.9 kDa, ADF3Kai-B A band of about 55.9 kDa and ADF3Kai-F of about 55.9 kDa) was observed, confirming that the target protein was expressed.

<Purification>
The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl (pH 7.4), and then cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged, and the resulting precipitate was lysed with 7.5 MUrea DB buffer (7.5 M urea, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) and stirred with a stirrer. After stirring, dialysis was performed with water using a dialysis tube (cellulose tube 36/32, Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, the water was removed with a freeze dryer, and the lyophilized powder was recovered. As a result of measuring the degree of purification of the target protein in the lyophilized powder as follows, the degree of purification of ADF3Kai, ADF3Kai-NRSH1, ADF3Kai-B, or ADF3Kai-F was 73.0%, 94.7%, and 95, respectively. 2% and 97.4%.

<Measurement of purity>
The degree of purification of the target protein in the lyophilized powder was measured by image analysis of the results of polyacrylamide gel electrophoresis of the lyophilized powder using Totallab (nonlinear dynamics ltd.). First, 4 mg of lyophilized powder was measured into an Eppendorf tube using a precision balance, and then 1 mL of 7.5 MUrea DB buffer (7.5 M urea, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0). Was added and dissolved sufficiently by vortexing for 20 minutes and standing at room temperature for 30 minutes.

≪Evaluation of solubility≫
(1) Preparation of spinning solution (dope solution) The lyophilized powder of ADF3Kai-F (molecular weight 60,000) obtained above was vacuum-dried for about 2 hours using a vacuum pump (ULVAC G-50DA), Water was completely removed from the lyophilized protein powder. Then, using a mortar, the protein from which moisture was removed was crushed as gently as possible to obtain a protein powder having a particle size of about 1 mm. Next, HFIP was added to the obtained protein powder and dissolved for about 19.5 hours while applying vibration with a shaker of 180 rpm at 25 ° C. to prepare a dope solution having a protein concentration of 7 mass%.

(5) Electrospinning Spinning was performed using the electrospinning apparatus 10 shown in FIG. The extrusion rate of the dope solution 2 in the microsyringe 1 was 2.4 ml / h, the voltage between the metal nozzle 3 and the metal roll 4 was 13.1 kV, and the distance was 75 mm. The metal nozzle 3 has an inner diameter of 0.32 mm and four nozzles. The rotation speed of the metal roll 4 (diameter 214 mm) was 170 rpm, and the winding speed was 114.2 m / min. Spinning was performed for 4 hours under these conditions. The sheet-like (nonwoven fabric) ultrafine fibers 7 wound up on the surface of the metal roll 4 were subjected to vacuum drying at 0.1 Pa, 35 ° C. for 1 hour to remove the solvent HFIP.

<Measurement of physical properties>
(A) Tensile test The obtained artificial polypeptide microfiber sheet (width 8 mm, length 20 mm, thickness 0.036 mm, mass per unit area (weight per unit area) 5.68 g / m ² ) at a pulling speed of 10 mm / min. The tensile test results were as shown in Table 1 below.

(B) Observation of the surface with a scanning electron microscope Using a scanning electron microscope (SEM), it observed with 5500 times, and measured the diameter of the single fiber. As a result, the average diameter of the side constant 5 (0.335 μm, 0.392 μm, 0.402 μm, 0.556 μm, 0.602 μm) was 0.457 μm. FIG. 3 shows a stress-displacement (strain) curve of the artificial polypeptide ultrafine fiber sheet obtained in Example 1.

(Example 2)
<Gene synthesis>
(1) Synthesis of ADF3Kai-Large Gene A PCR reaction was carried out using ADF3Kai as a template using a T7 promoter primer (SEQ ID NO: 18) and Rep Xba I primer (SEQ ID NO: 19), and the 5 ′ half of the ADF3Kai gene sequence The sequence (hereinafter referred to as “sequence A”) was amplified, and the fragment was subjected to restriction enzyme treatment with Nde I and Xba I in advance using a Mighty Cloning Kit (manufactured by Takara Bio Inc.). Recombined. Similarly, PCR reaction was performed using ADF3Kai as a template and Xba I Rep primer (SEQ ID NO: 21) and T7 terminator primer (SEQ ID NO: 20), and the 3 ′ half sequence (hereinafter referred to as sequence B) in the gene sequence of ADF3Kai. The fragment was recombined into a pUC118 vector previously treated with Xba I and EcoR I using a Mighty Cloning Kit (Takara Bio Inc.). The pUC118 vector into which the sequence A was introduced was treated with Nde I and Xba I, and the pUC118 vector into which the sequence B was introduced was treated with restriction enzymes Xba I and EcoR I, respectively. The DNA fragment was purified. DNA fragment A, DNA fragment B, and pET-22b (+) previously treated with Nde I and EcoR I were ligated and transformed into E. coli DH5α. After confirming the insertion of the target DNA fragment by colony PCR using the T7 promoter primer and T7 terminator primer, a plasmid was extracted from the colony from which a band in the vicinity of the target size (about 3.6 kbp) was obtained, and 3130xl Genetic Analyzer ( The entire base sequence was confirmed by a sequence reaction using Applied Biosystems). As a result, the construction of the ADF3Kai-Large gene consisting of the base sequence shown in SEQ ID NO: 16 was confirmed. The amino acid sequence of ADF3Kai-Large is as shown in SEQ ID NO: 4.

(2) Synthesis of ADF3Kai-Large-NRSH1 Gene By site-directed mutagenesis using PrimeStar Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) using pET22b (+) vector introduced with ADF3Kai-Large gene as a template. The codon GGC corresponding to the 1155th amino acid residue Gly of the amino acid sequence of ADF3Kai-Large (SEQ ID NO: 4) is mutated to the stop codon TAA, and the ADF3Kai-Large-NRSH1 gene consisting of the base sequence shown in SEQ ID NO: 17 is Built on pET22b (+). The accuracy of the introduction of the mutation was confirmed by a sequencing reaction using 3130xl Genetic Analyzer (Applied Biosystems). The amino acid sequence of ADF3Kai-Large-NRSH1 is as shown in SEQ ID NO: 15.

  Using the ADF3Kai-Large-NRSH1 (molecular weight of about 100,000) obtained as described above, purification was performed in the same manner as in Example 1, and the dope solution was prepared in the same manner as in Example except that the dope concentration was 4% by mass. Created and electrospun.

<Measurement of physical properties>
(A) Tensile test The obtained artificial polypeptide ultrafine fiber sheet (width 8 mm, length 20 mm, thickness 0.024 mm, mass per unit area (weight) 3.58 g / m ² ) at a tensile speed of 10 mm / min. The tensile test results were as shown in Table 2 below.

(B) Observation with a scanning electron microscope Using a scanning electron microscope (SEM), it observed at 5500 times, and measured the diameter of the single fiber. As a result, the average diameter of the side constant 5 (0.259 μm, 0.230 μm, 0.257 μm, 0.181 μm, 0.181 μm) was 0.22 μm. FIG. 4 shows a stress-displacement (strain) curve of the artificial polypeptide ultrafine fiber sheet obtained in Example 2, and FIG. 5 shows a SEM photograph.

  The artificial polypeptide fiber of the present invention can be suitably used for resin or metal reinforcing fibers, composite materials, injection molding and the like. The application can be applied to transportation equipment members such as automobiles and reinforcing fibers for tires. In addition, the present invention can be applied to woven fabrics, knitted fabrics, braided fabrics, nonwoven fabrics and the like.

DESCRIPTION OF SYMBOLS 1 Micro syringe 2 Dope liquid 3 Cap nozzle 4 Metal roll 5 Power supply 6 Fibrous material 7,9 Sheet-like (nonwoven fabric) extra fine fiber 8 Wire net 10,10 Electrospinning device

SEQ ID NOs: 1-10, 15 amino acid sequences SEQ ID NOs: 11-14, 16, 17 nucleotide sequences SEQ ID NOs: 18-21 primer sequences

Claims (4)

Spinning solution containing a polypeptide derived from nature spider silk protein (spider silk proteins) the method for manufacturing a human forming polypeptide microfibrous sheet that is obtained by electrospinning,
Dissolving the polypeptide in a solvent to form a spinning solution;
A step of forming fibers by discharging the spinning solution from a base to which voltage is applied by electrospinning ;
Including desorbing the solvent from the fiber by drying under reduced pressure ,
The average diameter of the single fibers of the ultrafine fibers is 1 μm or less, and the single fiber diameter of the ultrafine fibers varies between 0.1 and 1 μm,
A method for producing an artificial polypeptide ultrafine fiber sheet, wherein the sheet is formed by entanglement of the ultrafine fiber itself . The method for producing an artificial polypeptide ultrafine fiber sheet according to claim 1 , wherein the artificial polypeptide ultrafine fiber is further chemically cross-linked at a part between polypeptide molecules contained in the fiber. The crosslinking is carbodiimide and Guru least one artificial polypeptide microfibrous sheet manufacturing method according to the cross-linking agent to claim 2 is a cross-linked reacted selected from glutaraldehyde. The method for producing an artificial polypeptide ultrafine fiber sheet according to any one of claims 1 to 3, wherein the fiber-forming substance discharged from the die is wound around a roller or collected on a wire mesh to form a sheet.