Polar Solvent Solution And Production Method Thereof

Abstract

A polar solvent solution of the present invention is a polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent. The solution has a moisture content of less than 5 mass % based on 100 mass % of the solution. A method for producing a polar solvent solution of the present invention includes changing a moisture content of the solution to adjust the viscosity of the solution. Further, another method for producing a polar solvent solution includes reducing a moisture content of the solution to increase the viscosity of the solution. Thus, the present invention provides a polar solvent solution that enables stable spinning and casting without lowering its viscosity when used as dopes for spinning, film, etc., and methods for producing the same.

Description

TECHNICAL FIELD

[0001] The present invention relates to a polar solvent solution that can keep its viscosity high and production methods thereof.

BACKGROUND ART

[0002] Polar solvents such as dimethylsulfoxide (DMSO) can dissolve substances such as polymers easily, so they are used for acrylic fiber polymerization and acrylic fiber spinning solutions, or as solvents for polyimide polymerization, etc. The inventors of the present invention have proposed application of the polar solvents as solvents of polypeptides such as spider silk proteins and silk proteins in Patent Documents 1 and 2.

PRIOR ART DOCUMENTS

Patent Documents

[0003] Patent Document 1: JP 5427322 B

[0004] Patent Document 2: JP 5584932 B

DISCLOSURE OF INVENTION

Problem to be Solved by the Invention

[0005] However, polar solvent solutions (e.g., solutions in which polypeptides such as spider silk proteins and silk proteins are dissolved in dimethylsulfoxide (DMSO)) may have reduced viscosities depending on how they are handled. The polar solvent solutions still have room for improvement in terms of performing stable spinning and casting when used as dopes for spinning, film, etc.

[0006] The present invention provides a polar solvent solution that enables stable spinning and casting without lowering its viscosity when used as dopes for spinning, film, etc., and methods for producing the same.

Means for Solving Problem

[0007] The present invention relates to a polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent. The solution has a moisture content (moisture percentage) of less than 5 mass % based on 100 mass % of the solution.

[0008] The present invention also relates to a method for producing a polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent. The method includes: changing a moisture content of the solution to adjust a viscosity of the solution.

[0009] The present invention also relates to a method for producing a polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent. The method includes: reducing a moisture content of the solution to increase a viscosity of the solution.

Effect of the Invention

[0010] The polar solvent solution of the present invention in which a solute containing a polyamino acid is dissolved in a polar solvent has a moisture content of less than 5 mass %. By doing so, it is possible to prevent the viscosity of the solution from lowering significantly, and thus spinning and casting are stabilized when the solution is used as dopes for spinning, film, etc. The production method of the present invention includes changing a moisture content of a polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent, so as to adjust a viscosity of the solution. By doing so, it is possible to obtain a polar solvent solution that enables stable spinning and casting. Moreover, the production method of the present invention includes reducing a moisture content of the solution to increase a viscosity of the solution. By doing so, it is possible to obtain a polar solvent solution that enables stable spinning and casting.

BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 is a graph showing a viscosity change with a temperature change in several examples and a comparative example of the present invention.

[0012] FIG. 2 is a graph showing a viscosity change when with or without humidity control and the concentration and temperature of protein are changed in other examples of the present invention.

[0013] FIG. 3 is a graph showing a change in moisture percentage when spider silk protein (powder) in an absolute dry state is exposed to an atmosphere.

DESCRIPTION OF THE INVENTION

[0014] The inventors of the present invention found that polyamino acid (particularly polypeptide) itself, as well as a polar solvent solution in which a solute containing the polyamino acid is dissolved in polar solvent, readily absorbs moisture and lowers its viscosity. To cope with this problem, the polar solvent solution of the present invention in which a solute containing a polyamino acid is dissolved in a polar solvent has a moisture content of less than 5 mass % (0 mass % or more and less than 5 mass %) based on 100 mass % of the solution. The moisture content of the polar solvent solution is preferably 0 mass % or more and 3 mass % or less, more preferably 0 mass % or more and 1.5 mass % or less. Within this range, the polyamino acid (particularly polypeptide) in a swollen state is dissolved in the polar solvent, and the viscosity of the polar solvent solution is maintained high. In a case where the moisture content is 5 mass % or more, the viscosity of the polar solvent solution decreases significantly, and spinnability and casting properties decrease accordingly when the solution is used as dopes for spinning, film, etc. In the present specification, the polar solvent solution is also called a dope. The following mainly describes a case of using polypeptide, which is an exemplary polyamino acid.

[0015] It is preferred that the polar solvent to be used in the present invention contain at least one aprotic polar solvent selected from the group consisting of (i) dimethylsulfoxide (DMSO), (ii) N,N-dimethylformamide (DMF), (iii) N,N-dimethylacetamide (DMA), and (iv) N-methyl-2-pyrrolidone (NMP). This is because the aprotic polar solvents can dissolve solutes containing polypeptides easily. Examples of the polar solvent to be used in the present invention other than the solvents containing the above-described aprotic polar solvents include solvents containing protic polar solvents such as hexafluoroisopropanol (HFIP), formic acid, and various kinds of alcohols (e.g., lower alcohols having 1 to 6 carbon atoms such as methanol, ethanol, and 2-propanol). As the polar solvent, the ratio of the total amount of the at least one aprotic polar solvent selected from the group consisting of (i)-(iv) described above is desirably 10 to 100 mass %, based on 100 mass % of the polar solvent as a whole. Within this range, the solubility of the solutes containing polypeptides can be enhanced.

[0016] Any solute that contains a polyamino acid (particularly polypeptide) can be used as the solute of the present invention. In the present specification, the polyamino acid refers to any polyamide compound polymerized through amide linkage between amino groups and carboxyl groups of amino acids. As the polyamino acid, the number of amino acids constituting the polyamide compound is preferably 15 or more, more preferably 20 or more, further preferably 30 or more, still further preferably 100 or more, and particularly preferably 500 or more, and preferably 6000 or less, more preferably 5000 or less, further preferably 3000 or less, and particularly preferably 2000 or less. The solute to be used in the present specification may be composed of, e.g., polyamino acid alone or contain one or more kinds of substances (e.g., carbonhydrate, synthetic resin) other than the polyamino acid in combination with the polypeptide. Moreover, the solute to be used in the present specification may be composed of, e.g., polypeptide alone or contain one or more kinds of substances (e.g., carbonhydrate, synthetic resin) other than the polypeptide in combination with the polypeptide. The polypeptide is preferably a structural protein, more preferably a structural protein including crystal regions. Such polypeptides can exhibit high strength and high toughness when formed into fibers, films, and the like. The structural protein refers to any protein involved in structures of living organisms, or any protein constituting structures created by living organisms. Examples of the structural protein include fibroin, sericin, collagen, keratin, elastin, and resillin.

[0017] The polypeptides are preferably fibroin such as spider silk proteins and silk proteins. Of these, spider silk proteins are particularly preferred because they have a high affinity for polar solvents and can be dissolved in the polar solvents easily.

[0018] When the polar solvent solution of the present invention is assumed to be 100 mass %, the concentration of the solute (e.g., spider silk protein) is desirably 2 to 50 mass %, further preferably 3 to 40 mass %, and particularly preferably 5 to 30 mass %. Within this range, the decrease or excessive increase of the viscosity of the polar solvent solution can be avoided effectively.

[0019] The polar solvent solution of the present invention, desirably in a state where undesired substances such as dust and bubbles have been removed, has a viscosity of preferably 10 to 100000 mPas, further preferably 15 to 20000 mPas, and particularly preferably 100 to 10000 mPas. The polar solvent solution within this viscosity range enables favorable wet spinning and film casting when used as dopes.

[0020] In the production method of the present invention, the viscosity of the polar solvent solution is adjusted by changing the moisture content of the polar solvent solution. Moreover, in the production method of the present invention, the viscosity of the polar solvent solution is increased by reducing the moisture content of the polar solvent solution. In these processes of manufacture, the moisture content of the polar solvent solution is adjusted to be preferably less than 5 mass %, more preferably 0 to 3 mass %, and further preferably 0 to 1.5 mass % based on 100 mass % of the solution. By doing so, it is possible to obtain a polar solvent solution that enables stable spinning and casting when used as dopes for spinning, film, etc.

[0021] In the production methods of the present invention, the adjustment for reducing the moisture content of the solution is achieved by, e.g., subjecting the solute or the solvent to heat drying or vacuum drying in advance, or adjusting the relative humidity of the atmosphere in at least one of the production and the storage of the solution, or vaporizing moisture of the produced solution by heating, or absorbing moisture using various kinds of moisture absorbents (moisture absorbent materials) such as zeolite, or combining these operations appropriately. Among the adjustment methods for reducing the moisture content of the solution described above, the method of drying the solute before dissolution in the solvent is favorably adopted. By doing so, the moisture content of the solution can be reduced more reliably and more efficiently. Moreover, in the case of changing the moisture content of the solution by adjusting the relative humidity of the atmosphere, it is advantageous that the relative humidity of the atmosphere in at least one of the production and the storage of the solution is kept at 1.3% RH or less. In order to keep the relative humidity of the atmosphere at 1.3% RH or less, it is preferred that processes such as the production and storage of the solution be carried out inside a dry room.

[0022] In the present invention, DMSO, which is suitably used as a polar solvent for dissolving a solute containing a polypeptide, is particularly advantageously used as, e.g., a solvent for dissolving a solute containing a spider silk protein. DMSO has a melting point of 18.4.degree. C. and a boiling point of 189.degree. C. DMSO has a much higher boiling point than hexafluoroisopropanol (HFIP) and hexafluroacetone (HFAc) having boiling points of 59.degree. C. and -26.5.degree. C., respectively, which have been used in conventional methods. Further, in view of the fact that DMSO has been used also in general industrial fields for acrylic fiber polymerization and acrylic fiber spinning solutions, and as solvents for polyimide polymerization, they are low cost substances with proven safety.

[0023] The spider silk proteins, which are exemplified as polypeptides to be contained in the solute of the present invention, are not limited particularly as long as they are natural spider silk proteins or proteins derived from or analogous to (hereinafter, simply referred to as "derived from") natural spider silk proteins. The proteins derived from natural spider silk proteins described herein are proteins having an amino acid sequence similar to or analogous to any of repetitive sequences of amino acids of natural spider silk proteins, examples of which includes variants, analogs, and derivatives of recombinant spider silk proteins and natural spider silk proteins. The spider silk proteins are preferably major dragline silk proteins produced in major ampullate glands of spiders or spider silk proteins derived therefrom, in terms of excellent tenacity. Examples of the major dragline silk proteins include major ampullate spidroins MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Araneus diadematus, etc.

[0024] The spider silk proteins may be minor dragline silk proteins produced in minor ampullate glands of spiders or spider silk proteins derived therefrom.

[0025] Examples of the minor dragline silk proteins include minor ampullate spidroins MiSp1 and MiSp2 derived from Nephila clavipes.

[0026] Other than these, the spider silk proteins may be flagelliform silk proteins produced in flagelliform glands of spiders or spider silk proteins derived therefrom. Examples of the flagelliform silk proteins include flagelliform silk proteins derived from Nephila clavipes, etc.

[0027] Examples of the spider silk proteins (polypeptides) derived from major dragline silk proteins include recombinant spider silk proteins containing two or more units of an amino acid sequence represented by the formula 1: REP1-REP2 (1), preferably recombinant spider silk proteins containing four or more units thereof, and more preferably recombinant spider silk proteins containing six or more units thereof. In the recombinant spider silk proteins, units of the amino acid sequence represented by the formula (1): REP1-REP2 (1) may be the same or different from each other.

[0028] In the formula (1), the REP1 represents a polyalanine region mainly constituted by alanine and expressed as (X1)p, and preferably the REP1 represents polyalanine. Here, p is not particularly limited, but preferably an integer of 2 to 20, more preferably an integer of 4 to 12. X1 represents alanine (Ala), serine (Ser), or glycine (Gly). The total number of alanine residues in the polyalanine region expressed as (X1)p is preferably 80% or more, more preferably 85% or more with respect to the total number of amino acid residues in the region. In the REP1, the number of alanine residues arranged in succession is preferably 2 or more, more preferably 3 or more, further preferably 4 or more, and particularly preferably 5 or more. Further, in the REP1, the number of alanine residues arranged in succession is preferably 20 or less, more preferably 16 or less, further preferably 12 or less, and particularly preferably 10 or less. In the formula (1), the REP2 is an amino acid sequence composed of 10 to 200 amino acid residues. The total number of glycine, serine, glutamine, proline and alanine residues contained in the amino acid sequence is 40% or more, preferably 50% or more, and more preferably 60% or more with respect to the total number of amino acid residues contained therein.

[0029] The REP1 corresponds to a crystal region in a fiber where a crystal 13 sheet is formed, and the REP2 corresponds to an amorphous region in a fiber where flexibility is high and most of the parts lack regular configurations. Further, the [REP1-REP2] corresponds to a repeating region (repetitive sequence) composed of the crystal region and the amorphous region, which is a characteristic sequence of dragline silk proteins.

[0030] Examples of the recombinant spider silk proteins containing two or more units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) are recombinant spider silk proteins derived from ADF3 having an amino acid sequence represented by any of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence obtained by the following mutation: in an amino acid sequence of ADF3 to the N-terminal of which has been added an amino acid sequence (SEQ ID NO: 4) composed of a start codon, His 10-tag and HRV3C Protease (Human rhinovirus 3C Protease) recognition site, 1.sup.st to 13.sup.th repetitive regions are about doubled and the translation ends at the 1154.sup.th amino acid residue. The amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence obtained by adding the amino acid sequence (SEQ ID NO: 4) composed of a start codon, His 10-tag and HRV3C Protease (Human rhinovirus 3C Protease) recognition site, to the N-terminal of a partial amino acid sequence of ADF3 (NCBI Genebank Accession No.: AAC47010, GI: 1263287) obtained from the NCBI database. The amino acid sequence represented by SEQ ID NO: 3 is an amino acid sequence obtained by the following mutation: in an amino acid sequence of ADF3 to the N-terminal of which has been added the amino acid sequence (SEQ ID NO: 4) composed of a start codon, His 10-tag and HRV3C Protease (Human rhinovirus 3C Protease) recognition site, 1.sup.st to 13.sup.th repetitive regions are about doubled. Further, the recombinant spider silk proteins containing two or more units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) may be spider silk proteins that are composed of an amino acid sequence represented by any of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in which one or more amino acids have been substituted, deleted, inserted and/or added and that have repeating regions composed of the crystal region and the amorphous region.

[0031] Examples of the spider silk proteins (polypeptides) derived from minor dragline silk proteins are recombinant spider silk proteins containing an amino acid sequence represented by the formula 2: REP3-REP4-REP5 (2). In the formula 2, the REP 3 indicates an amino acid sequence represented by (Gly-Gly-Z)m, the REP4 indicates an amino acid sequence represented by (Gly-Ala)l, and the REP5 indicates an amino acid sequence represented by (Ala)r. In the REP3, Z indicates any one of amino acids, particularly, it is preferably an amino acid selected from the group consisting of Ala, Tyr and Gin. Further, in the REP3, m is preferably 1 to 4. In the REP4, l is preferably 0 to 4. In the REP 5, r is preferably 1 to 6.

[0032] Among spider silks, the minor dragline silk is wound spirally from the center of a spider net, and used as a reinforcement of the net and a yarn to wrap a captured prey. The minor dragline silk is inferior to the major dragline silk in tensile strength, but is known to have high stretchability. The reason for this is considered to be as follows: in the minor dragline silk, since many crystal regions are composed of regions where glycine and alanine are arranged alternately in succession, the hydrogen bonds of the crystal regions weaken easily as compared with the major dragline silk whose crystal regions are composed only of alanine.

[0033] Examples of the recombinant spider silk proteins (polypeptides) derived from flagelliform silk proteins include recombinant spider silk proteins containing an amino acid sequence represented by the formula 3: REP6 (3). In the formula 3, the REP 6 indicates an amino acid sequence represented by (U1)n or (U2)n. In the REP6, U1 indicates an amino acid sequence represented by Gly-Pro-Gly-X-X (SEQ ID NO: 12), and U2 indicates an amino acid sequence represented by Gly-Pro-Gly-Gly-X (SEQ ID NO: 13). In the U1 and U2, X indicates any one of amino acids, particularly, it is preferably an amino acid selected from the group consisting of Ala, Ser, Tyr, Gin, Val, Leu, and Ile, more preferably an amino acid selected from the group consisting of Ala, Ser, Tyr, Gin, and Val. A plurality of X may be the same or different from each other. In the REP6, n indicates a number of 4 or larger, preferably 10 or larger, and more preferably 20 or larger.

[0034] Among spider silks, the flagelliform silk does not have crystal regions but has repeating regions composed of the amorphous region, which is a major characteristic of the flagelliform silk. It is considered that since the major dragline silk and the like have repeating regions composed of the crystal region and the amorphous region, they have both high stress and stretchability. Meanwhile, regarding the flagelliform silk, the stress is inferior to that of the major dragline silk but the stretchability is high. The reason for this is considered to be that the flagelliform silk is composed mostly of the amorphous region.

[0035] The recombinant spider silk proteins (polypeptides) can be produced using a host that has been transformed by an expression vector containing a gene encoding a natural spider silk protein subjected to recombination. A method for producing a gene is not limited particularly, and it may be produced by amplifying a gene encoding a natural spider silk protein from a cell derived from spiders by a polymerase chain reaction (PCR) or the like, and cloning it, or may be synthesized chemically. A method for chemically synthesizing a gene also is not limited particularly, and it can be synthesized as follows, for example: based on information of amino acid sequences of natural spider silk proteins obtained from the NCBI web database or the like, oligonucleotides that have been synthesized automatically with AKTA oligopilot plus 10/100 (GE Healthcare Japan Corporation) are linked by PCR or the like. At this time, in order to facilitate purification and observation of protein, a gene may be synthesized that encodes a protein having the above-described amino acid sequence to the N-terminal of which has been added an amino acid sequence composed of a start codon and His 10-tag. Examples of the expression vector include a plasmid, a phage, a virus and the like that can express protein based on a DNA sequence. The plasmid-type expression vector is not limited particularly as long as it allows a target gene to be expressed in a host cell and it can amplify itself. For example, in the case of using Escherichia coli Rosetta (DE3) as a host, a pET22b(+) plasmid vector, a pCold plasmid vector and the like can be used. Among these, in terms of productivity of protein, it is preferable to use the pET22b(+) plasmid vector. Examples of the host include animal cells, plant cells, microbes, etc.

EXAMPLES

[0036] Hereinafter, the present invention will be described in further detail by way of examples. Note that the present invention is not limited to the following examples.

[0037] <Various Measurement Methods>

[0038] (1) Viscosity: The viscosities of polar solvent solutions (dopes) were measured using an EMS viscometer (EMS-01S) manufactured by Kyoto Electronics Manufacturing Co., Ltd.

[0039] (2) Relative humidity: The temperature and the dew-point temperature of an experiment environment were measured to calculate the relative humidity of the environment using a known calculation.

[0040] (3) Moisture percentage of dope: The moisture percentages of dopes were measured using a Hybrid Karl Fischer Moisture Titrator (MKH-700) manufactured by Kyoto Electronics Manufacturing Co., Ltd.

Examples 1-4, Comparative Example 1

[0041] 1. Preparation of Spider Silk Proteins

[0042] <Gene Synthesis>

[0043] (1) Gene Synthesis of ADF3Kai

[0044] A partial amino acid sequence of ADF3 (GI: 1263287), which is one of two principal dragline silk proteins of Araneus diadematus, was obtained from the NCBI web database, and synthesis of a gene encoding an amino acid sequence (SEQ ID NO: 2) was outsourced to GenScript, Inc. The amino acid sequence (SEQ ID NO: 2) is an amino acid sequence obtained by adding an amino acid sequence (SEQ ID NO: 4) composed of a start codon, His 10-tag and HRV3C Protease (Human rhinovirus 3C Protease) recognition site, to the N-terminal of said partial amino acid sequence of ADF3. As a result, a pUC57 vector to which a gene of ADF3Kai having a base sequence represented by SEQ ID NO: 5 had been introduced was obtained (having an Nde I site immediately upstream of 5' terminal of the gene and an Xba I site immediately downstream of 5' terminal thereof). Thereafter, the gene was subjected to a restriction enzyme treatment with Nde I and EcoR I, and recombined into a pET22b(+) expression vector.

[0045] (2) Gene Synthesis of ADF3Kai-Large

[0046] The half of the gene sequence of ADF3Kai on the 5' side (hereinafter, referred to as a sequence A) was amplified by the PCR reaction using ADF3Kai as a template, and a T7 promoter primer (SEQ ID NO: 8) and a Rep Xba I primer (SEQ ID NO: 9). The obtained DNA fragment of the sequence A was recombined into a pUC118 vector that had been subjected to the restriction enzyme treatment with Nde I and Xba I in advance using a Mighty Cloning Kit (manufactured by TAKARA BIO INC.). Similarly, the half of the gene sequence of ADF3Kai on the 3' side (hereinafter, referred to as a sequence B) was amplified by the PCR reaction using ADF3Kai as a template, and an Xba I Rep primer (SEQ ID NO: 10) and a T7 terminator primer (SEQ ID NO: 11). The obtained DNA fragment of the sequence B was recombined into a pUC118 vector that had been subjected to the restriction enzyme treatment with Xba I and EcoR I in advance using the Mighty Cloning Kit (manufactured by TAKARA BIO INC.). The pUC118 vector to which the sequence A had been introduced and the pUC118 vector to which the sequence B had been introduced were subjected to the restriction enzyme treatment with Nde I, Xba I and Xba I, EcoR I, respectively, and target DNA fragments of the sequences A and B were purified by gel cut. The DNA fragments A, B and the pET22b(+) that had been subjected to the restriction enzyme treatment with Nde I and EcoR I in advance were subjected to a ligation reaction and transformed into Escherichia coli DH5a. After confirmation of the insertion of the target DNA fragments by a colony PCR using a T7 promoter primer and a T7 terminator primer, plasmid was extracted from a colony where a target band size (3.6 kbp) was obtained, and the entire base sequence was checked by a sequence reaction using a 3130.times.1 Genetic Analyzer (Applied Biosystems). Consequently, the construction of a gene of ADF3Kai-Large represented by SEQ ID NO: 6 was confirmed. The amino acid sequence of ADF3Kai-Large is as represented by SEQ ID NO: 3.

[0047] (3) Gene Synthesis of ADF3Kai-Large-NRSH1

[0048] With a pET22b(+) vector to which the gene of ADF3Kai-Large obtained above had been introduced used as a template, through site-directed mutagenesis using a PrimeSTAR Mutagenesis Basal Kit (manufactured by TAKARA BIO INC.), a codon GGC corresponding to the 1155.sup.th amino acid residue, i.e., glycine (Gly), in the amino acid sequence of ADF3Kai-Large (SEQ ID NO: 3) was mutated into a stop codon TAA, and a gene of ADF3Kai-Large-NRSH1 represented by SEQ ID NO: 7 was constructed on the pET22b(+). The accuracy of the introduction of the mutation was checked by the sequence reaction using the 3130.times.1 Genetic Analyzer (Applied Biosystems). The amino acid sequence of ADF3Kai-Large-NHSH1 is as represented by SEQ ID NO: 1.

[0049] <Expression of Protein>

[0050] The pET22b(+) expression vector containing the gene sequence of ADF3Kai-Large-NHSH1 was transformed into Escherichia coli Rosetta (DE3). The obtained single colony was incubated for 15 hours in 2 ml of an LB culture medium containing ampicillin. Thereafter, 1.4 ml of the culture solution was added to 140 ml of an LB culture medium containing ampicillin, and incubated to an OD.sub.600 of 3.5 under the conditions of 37.degree. C. and 200 rpm. Next, the culture solution with the OD.sub.600 of 3.5 was added to 7 L of a 2.times.YT culture medium containing ampicillin, together with 140 ml of 50% glucose, and incubated further to the OD.sub.600 of 4.0. Thereafter, isopropyl-6-thiogalactopyranoside (IPTG) was added to the obtained culture solution with the OD.sub.600 of 4.0 so that the final concentration would be 0.5 mM, thereby inducing the expression of protein. After a lapse of two hours from the addition of IPTG, the culture solution was centrifuged and bacterial cells were collected. Protein solutions prepared from the culture solution before the addition of IPTG and after the addition of IPTG were each electrophoresed in a polyacrylamide gel. Consequently, a target band size (about 101.1 kDa) was observed with the addition of IPTG, and the expression of the target protein was confirmed.

[0051] Purification

[0052] (1) About 50 g of bacteria cells of the Escherichia coli expressing the ADF3Kai-Large-NRSH1 protein and 300 ml of a buffer solution AI (20 mM Tris-HCl, pH 7.4) were placed in a centrifuge tube (1000 ml). After dispersing the bacteria cells with a mixer ("T18 basic ULTRA TURRAX" manufactured by IKA, level 2), the dispersion was centrifuged (11,000 g, 10 minutes, room temperature) with a centrifuge ("Model 7000" manufactured by Kubota Corporation), and a supernatant was discarded.

[0053] (2) To a precipitate (bacteria cells) obtained by the centrifugation, 300 ml of the buffer solution AI and 3 ml of 0.1 M PMSF (dissolved by isopropanol) were added. After dispersing the precipitate for 3 minutes with the mixer (level 2) manufactured by IKA, the bacteria cells were disrupted repeatedly for three times using a high-pressure homogenizer ("Panda Plus 2000" manufactured by GEA Niro Soavi).

[0054] (3) To the disrupted bacterial cells, 300 ml of a buffer solution B (50 mM Tris-HCL, 100 mM NaCl, pH 7.0) containing 3 w/v % of SDS was added. After dispersing well the bacterial cells with the mixer (level 2) manufactured by IKA, the dispersion was stirred for 60 minutes with a shaker (manufactured by TAITEC CORPORATION, 200 rpm, 37.degree. C.). Thereafter, the stirred dispersion was centrifuged (11,000 g, 30 minutes, room temperature) with the centrifuge manufactured by Kubota Corporation, and a supernatant was discarded, whereby SDS washing granules (precipitate) were obtained.

[0055] (4) The SDS washing granules were suspended in a DMSO solution containing LM lithium chloride so that the concentration would be 100 mg/ml, and heat-treated for 1 hour at 80.degree. C. Thereafter, the heated suspension was centrifuged (11,000 g, 30 minutes, room temperature) with the centrifuge manufactured by Kubota Corporation, and the supernatant was collected.

[0056] (5) Ethanol in an amount three times greater than that of the collected supernatant was prepared. The collected supernatant was added to the ethanol, and left to stand still for 1 hour at room temperature. Thereafter, the resultant was centrifuged (11,000 g, 30 minutes, room temperature) with the centrifuge manufactured by Kubota Corporation to collect aggregated protein. Next, a process of washing aggregated protein using pure water and a process of collecting aggregated protein by centrifugation were repeated three times, and then moisture was removed by a freeze dryer to collect freeze-dried powder. The purification degree of the target protein ADF3Kai-Large-NRSH1 (about 56.1 kDa) in the obtained freeze-dried powder was checked by analyzing images of the results of polyacrylamide gel electrophoresis (CBB staining) of said protein powder using Totallab (nonlinear dynamics Ltd.). As a result, the purification degree of ADF3Kai-Large-NRSH1 was about 85%.

[0057] 2. Adjustment of Dopes and Viscosity Measurement

[0058] The spider silk protein (powder) obtained above was subjected to vacuum drying (bone dry), and the spider silk protein in the absolute dry state was added to five DMSO solvents of a predetermined amount prepared beforehand so that the concentration of the protein of the respective solvents would be 15 mass %. Different amounts of pure water were added and mixed into four of the five DMSO solvents containing the spider silk protein to prepare five kinds of dopes having different moisture contents (moisture percentages) as indicated in Table 1 below. The dopes with a moisture content of 0 mass %, 0.75 mass %, 1.5 mass %, and 3 mass % are dopes of Examples 1, 2, 3, and 4, respectively. The dope with a moisture content of 5 mass % is a dope of Comparative Example 1. In the preparation of the five kinds of dopes of Examples 1-4 and Comparative Example 1, the spider silk protein was dissolved in the solvents for 5 hours using a shaker, and then dust and bubbles were removed from the solvents. This process was all performed in a dry room at a relative humidity of 1.3% RH or less. The storage was also in a dry room at a relative humidity of 1.3% RH or less. The viscosity change with temperature was tested for the dopes of Examples 1-4 and the dope of Comparative Example 1. Table 1 below and FIG. 1 show the results.

TABLE-US-00001 TABLE 1 Viscosity (mPa s) Temperature (.degree. C.) 70 60 50 40 30 25 Ex. 1 H.sub.2O, 0 mass % 82 110 153 221 333 434 Ex. 2 H.sub.2O, 0.75 mass % 67 89 124 193 306 393 Ex. 3 H.sub.2O, 1.5 mass % 56 73 100 150 232 299 Ex. 4 H.sub.2O, 3 mass % 49 63 89 132 199 261 Comp. H.sub.2O, 5 mass % 23 30 39 53 76 92 Ex. 1 *Ex.: Example, Comp. Ex.: Comparative Example

[0059] As is clear from Table 1 and FIG. 1, the dopes of Examples 1-4 with a moisture content of less than 5 mass % had high viscosities regardless of the temperature, and the viscosity rise in accordance with the temperature drop was significant, as compared with the dope of Comparative Example 1 with a moisture content of 5 mass %. Further, as to the dopes of Examples 1-4, the viscosity was high as the moisture content was low, i.e., the dope of Example 1 with a moisture content of 0 mass % had the highest viscosity. Moreover, at a temperature of 25.degree. C., which is close to room temperature, the viscosities of the dopes of Examples 1-4 were much higher than the viscosity of the dope of Comparative Example 1, specifically, they were 2.8 to 4.7 times the viscosity of the dope of Comparative Example 1. These results clearly indicate that the viscosities of the dopes can be increased by adjusting the moisture content of the dopes to be less than 5 mass %. It was also confirmed that spinning and casting can be stabilized with the dopes of Examples 1-4.

Examples 5-9

[0060] This experiment was carried out to examine the viscosity change when with or without humidity control and the concentration and temperature of spider silk protein were changed. First, the spider silk protein (powder) obtained above was subjected to vacuum drying (bone dry), and the spider silk protein (powder) in the absolute dry state (moisture content: 0 mass %) was dissolved in DMSO solvents at concentrations indicated in Table 2 below in a dry room at a relative humidity of the atmosphere of 1.3% RH or less, so as to produce four kinds of dopes (Examples 5-8) having different concentrations of the spider silk protein. Then, the four kinds of dopes of Examples 5-8 were stored in a dry room at a relative humidity of 1.3% RH or less for 24 hours. Further, the spider silk protein (powder) in the absolute dry state was dissolved in a DMSO solvent at a concentration of 22.0 mass % in a general laboratory (in the atmosphere) without humidity control to produce a dope of Example 9. The dope of Example 9 was stored in a general laboratory without humidity control for 24 hours. The respective conditions of the experiment are shown in Table 2 below. A relationship between the temperature and the viscosity of the dopes of Examples 5-9 was examined. FIG. 2 shows the results.

TABLE-US-00002 TABLE 2 Ex. 5 Ex. 6 Ex. 7 Ex. 8 Ex. 9 Protein concentration (mass %) 19.5 20.0 20.5 21.5 22.0 With or without adjustment in With With With With Without dry room With or without storage in With With With With Without dry room

[0061] As indicated in FIG. 2, the relationship between the temperature and the viscosity of the dope of Example 9 (produced and stored in an environment without humidity control and having a protein concentration of 22.0 mass %) was substantially the same as that of the dope of Example 7 (produced and stored in the dry room and having a protein concentration of 20.5 mass %). The reason for this is considered to be that the moisture in the atmosphere was mixed into the dope of Example 9 during the production and storage. These results indicate that the viscosities of the dopes can be kept high by lowering the relative humidity of the atmosphere in the production and storage of the dopes to prevent the dopes from absorbing moisture.

[0062] (Reference Test 1)

[0063] The spider silk protein (powder) obtained above was subjected to vacuum drying (bone dry), and the spider silk protein (powder) in the absolute dry state was exposed to an atmosphere at a temperature of 25.degree. C. and a relative humidity of 72% RH to examine a change in moisture percentage. FIG. 3 shows the results. As is clear from FIG. 3, the moisture percentage of the spider silk protein (powder) in the absolute dry state reached about 13 mass % (equilibrium moisture regain) in about 20 minutes. This test result indicates that it is important for the spider silk protein (powder) to be subjected to vacuum drying to prepare dopes. Without the vacuum drying, the moisture adsorbed to the spider silk protein (powder) during the storage under the room temperature or an environment with high relative humidity is mixed into dopes directly.

[0064] (Reference Test 2)

[0065] The following test was performed to confirm that the lowering of the viscosity of the dope due to the mixing of moisture into the dope was not simply attributed to the dilution of the dope with moisture. First, a dishwashing detergent having almost the same viscosity as that of the dope of Example 1 at 50.degree. C. was prepared. Next, moisture was added to the dishwashing detergent so that the moisture content would be 3 mass % based on 100 mass % of the dishwashing detergent. Then, the viscosity of the dishwashing detergent at 50.degree. C. was measured to determine a rate of change of viscosity before and after addition of moisture.

[0066] As a result, the viscosity of the dishwashing detergent at 50.degree. C. before addition of moisture was 143 mPas, and the viscosity of the dishwashing detergent at 50.degree. C. after addition of moisture was 133 mPas. A lowering rate of the viscosity of the dishwashing detergent at 50.degree. C. due to addition of moisture was 7%. Meanwhile, the viscosity of the dope of Example 1 at 50.degree. C. was 153 mPas, and the viscosity of the dope of Example 4 at 50.degree. C. with a moisture content of 3 mass % by addition of moisture was 89 mPas. A lowering rate of the viscosity of the dope containing the spider silk protein at 50.degree. C. due to addition of moisture was 42%. It was clearly recognized from these results that the lowering of the viscosity of the polar solvent solution containing polypeptide (e.g., spider silk protein) due to the mixing of moisture into the solution was not simply attributed to the dilution with moisture.

[0067] The following are considered as the reasons for the significant lowering of the viscosity of the polar solvent solution of polypeptide due to the mixing (inclusion) of moisture into the solution. Amino acids constituting molecules of protein (polypeptide) have various side chains. When water molecules enter the polar solvent solution of protein, hydrogen bonds are formed between the side chains of the protein molecules, and the protein molecules agglomerate. This decreases the solubility of the protein, and lowers the viscosity of the polar solvent solution of protein. Therefore, by removing water molecules from the polar solvent solution of protein, the agglomeration of the protein molecules can be avoided, and the solubility of the protein into the polar solvent solution can be enhanced, resulting in an increase in the viscosity of the polar solvent solution.

INDUSTRIAL APPLICABILITY

[0068] The polar solvent solution of the present invention is useful for wet spinning, film casting, gels, particles, mesh materials, and various types of moldings.

Sequence Listing Free Text

[0069] SEQ ID NOS: 1-4, 12, 13 amino acid sequences

[0070] SEQ ID NOS: 5-7 base sequences

[0071] SEQ ID NOS: 8-11 primer sequences

Sequence CWU 1

1

1311154PRTArtificialADF3Kai-Large-NRSH1 1Met His His His His His His His His His His Ser Ser Gly Ser Ser 1 5 10 15 Leu Glu Val Leu Phe Gln Gly Pro Ala Arg Ala Gly Ser Gly Gln Gln 20 25 30 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 50 55 60 Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln 65 70 75 80 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 85 90 95 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 115 120 125 Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln 130 135 140 Gly Pro Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 145 150 155 160 Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly 165 170 175 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 180 185 190 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln 195 200 205 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 210 215 220 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 225 230 235 240 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 245 250 255 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 260 265 270 Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 275 280 285 Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly 290 295 300 Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 305 310 315 320 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 325 330 335 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 340 345 350 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 355 360 365 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 370 375 380 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 385 390 395 400 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 405 410 415 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 420 425 430 Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly 435 440 445 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 450 455 460 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 465 470 475 480 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly 485 490 495 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 500 505 510 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 515 520 525 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser 530 535 540 Val Ser Arg Ala Arg Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln 545 550 555 560 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 565 570 575 Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly 580 585 590 Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 595 600 605 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly 610 615 620 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 625 630 635 640 Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly 645 650 655 Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln 660 665 670 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 675 680 685 Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 690 695 700 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 705 710 715 720 Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 725 730 735 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 740 745 750 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 755 760 765 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 770 775 780 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly 785 790 795 800 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 805 810 815 Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln 820 825 830 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro 835 840 845 Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser 850 855 860 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 865 870 875 880 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 885 890 895 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 900 905 910 Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 915 920 925 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 930 935 940 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly 945 950 955 960 Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly 965 970 975 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 980 985 990 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 995 1000 1005 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 1010 1015 1020 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 1025 1030 1035 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 1040 1045 1050 Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser Val 1055 1060 1065 Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala 1070 1075 1080 Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser 1085 1090 1095 Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala 1100 1105 1110 Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala 1115 1120 1125 Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu 1130 1135 1140 Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu 1145 1150 2660PRTArtificialADF3Kai 2Met His His His His His His His His His His Ser Ser Gly Ser Ser 1 5 10 15 Leu Glu Val Leu Phe Gln Gly Pro Ala Arg Ala Gly Ser Gly Gln Gln 20 25 30 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 50 55 60 Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln 65 70 75 80 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 85 90 95 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 115 120 125 Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln 130 135 140 Gly Pro Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 145 150 155 160 Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly 165 170 175 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 180 185 190 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln 195 200 205 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 210 215 220 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 225 230 235 240 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 245 250 255 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 260 265 270 Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 275 280 285 Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly 290 295 300 Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 305 310 315 320 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 325 330 335 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 340 345 350 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 355 360 365 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 370 375 380 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 385 390 395 400 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 405 410 415 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 420 425 430 Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly 435 440 445 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 450 455 460 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 465 470 475 480 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly 485 490 495 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 500 505 510 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 515 520 525 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser 530 535 540 Val Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala 545 550 555 560 Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser 565 570 575 Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu 580 585 590 Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro 595 600 605 Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val 610 615 620 Ser Ala Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn 625 630 635 640 Tyr Gly Ala Ser Ala Gln Tyr Thr Gln Met Val Gly Gln Ser Val Ala 645 650 655 Gln Ala Leu Ala 660 31183PRTArtificialADF3Kai-Large 3Met His His His His His His His His His His Ser Ser Gly Ser Ser 1 5 10 15 Leu Glu Val Leu Phe Gln Gly Pro Ala Arg Ala Gly Ser Gly Gln Gln 20 25 30 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 50 55 60 Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln 65 70 75 80 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 85 90 95 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 115 120 125 Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln 130 135 140 Gly Pro Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 145 150 155 160 Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly 165 170 175 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 180 185 190 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln 195 200 205 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 210 215 220 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 225 230 235 240 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 245 250 255 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 260 265 270 Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 275 280 285 Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly 290 295 300 Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 305 310 315 320 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 325 330 335 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 340 345 350 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 355 360 365 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 370 375 380 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 385 390 395 400 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 405 410 415 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 420 425 430 Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly 435 440 445 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 450 455 460 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln

Gly Pro Gly 465 470 475 480 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly 485 490 495 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 500 505 510 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 515 520 525 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser 530 535 540 Val Ser Arg Ala Arg Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln 545 550 555 560 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 565 570 575 Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly 580 585 590 Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 595 600 605 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly 610 615 620 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 625 630 635 640 Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly 645 650 655 Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln 660 665 670 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 675 680 685 Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 690 695 700 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 705 710 715 720 Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 725 730 735 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 740 745 750 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 755 760 765 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 770 775 780 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly 785 790 795 800 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 805 810 815 Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln 820 825 830 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro 835 840 845 Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser 850 855 860 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 865 870 875 880 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 885 890 895 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 900 905 910 Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 915 920 925 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 930 935 940 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly 945 950 955 960 Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly 965 970 975 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 980 985 990 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 995 1000 1005 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 1010 1015 1020 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 1025 1030 1035 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 1040 1045 1050 Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser Val 1055 1060 1065 Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala 1070 1075 1080 Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser 1085 1090 1095 Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala 1100 1105 1110 Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala 1115 1120 1125 Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu 1130 1135 1140 Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu Gly Ser Ser Ser 1145 1150 1155 Ile Gly Gln Ile Asn Tyr Gly Ala Ser Ala Gln Tyr Thr Gln Met 1160 1165 1170 Val Gly Gln Ser Val Ala Gln Ala Leu Ala 1175 1180 424PRTArtificialHis tag and start codon 4Met His His His His His His His His His His Ser Ser Gly Ser Ser 1 5 10 15 Leu Glu Val Leu Phe Gln Gly Pro 20 51983DNAArtificialADF3Kai 5atgcatcacc atcatcatca tcaccaccac cattcctcgg gctcatcctt ggaagtgtta 60tttcaaggac cagcacgagc cggttcggga caacaagggc ctggccagca gggcccaggt 120caacaagggc caggacagca gggtccttat gggcccggcg caagcgcagc agctgcggcc 180gctggtggct atggtcctgg ctccggtcaa cagggccctt cgcaacaagg tcccgggcag 240caaggtcctg gtggccaggg tccctacggg ccgggggcga gtgcggcagc agccgctgca 300ggcggttatg gtccaggaag cggacagcaa ggtccgggag gtcaaggtcc gtatggccca 360ggctctagcg cggctgccgc tgccgcgggt ggcaacggac cagggagcgg acaacagggc 420gcgggacaac agggtccagg acagcaaggc ccaggggcgt cggcggctgc agcggcggcc 480ggaggctatg gacccggctc aggacaacag ggaccgggtc aacaaggacc cggtggccaa 540ggcccctatg gcccgggcgc cagcgcggcc gcagccgccg cgggcgggta cggccccggt 600agcggccagg gaccaggtca gcaggggcca ggaggtcagg gcccatacgg tccgggcgca 660tccgcggcgg cggcagcggc aggtggctac ggtcccggaa gcggccaaca ggggccaggg 720caacaaggac caggacaaca aggtcctggg ggccaaggac cgtatggacc aggagcatca 780gctgcagccg cggcagctgg cggttacggt ccaggctacg gccagcaggg tccgggtcag 840cagggaccgg gaggccaggg gccttatggc cctggcgctt ccgcagccag tgccgcttct 900ggaggatacg ggccgggaag cggtcagcaa ggccctggcc aacaaggacc tggaggccaa 960gggccctacg gcccaggagc ctcggcagcc gcagctgccg caggtgggta tgggccaggt 1020agcgggcaac aagggccggg tcagcaagga ccggggcaac agggacctgg gcagcaagga 1080cccgggggtc aaggcccgta cggacctggt gcgtctgcag ctgctgctgc ggctggtgga 1140tatggtccgg gatcggggca gcagggtccc ggtcagcagg gccctggtca gcaagggcca 1200ggccaacagg gacccggaca acaaggcccg ggtcaacagg gtcctggaca gcaggggccg 1260ggccaacaag gccctgggca acagggtccg gggggacagg gggcctatgg gcctggcgca 1320tctgccgccg ctggcgcagc cggtgggtac gggcctgggt caggtcaaca ggggcctggt 1380caacaaggcc ccgggcaaca gggccccggc cagcaaggtc cagggcagca gggcccggga 1440cagcaagggc ctggacaaca ggggcccgga cagcagggac cttacgggcc cggtgcgagc 1500gcagcggccg ccgccgcagg gggatatggc cccggatcgg gccagcaggg accaggccag 1560caaggacctg gccaacaggg cccggggggt caggggccgt atggtcccgg cgctgcaagt 1620gctgcagtgt ccgttggagg ttacggccct cagtcttcgt ctgttccggt ggcgtccgca 1680gttgcgagta gactgtcttc acctgctgct tcatcgcgag tatcgagcgc tgtttcgtct 1740cttgtctcgt cgggtcccac gaaacatgcc gccctttcaa atacgatttc atctgtagtg 1800tcccaagtta gtgcaagtaa cccggggtta tccggatgcg acgttctcgt tcaggcactc 1860ctagaagtag tatccgcgtt ggtgagcatc ttaggcagct cctcgatagg tcaaataaac 1920tatggtgctt cagcccagta tacacagatg gtgggacaga gcgtcgcgca ggcattggct 1980taa 198363552DNAArtificialADF3Kai-Large 6atgcatcacc atcatcatca tcaccaccac cattcctcgg gctcatcctt ggaagtgtta 60tttcaaggac cagcacgagc cggttcggga caacaagggc ctggccagca gggcccaggt 120caacaagggc caggacagca gggtccttat gggcccggcg caagcgcagc agctgcggcc 180gctggtggct atggtcctgg ctccggtcaa cagggccctt cgcaacaagg tcccgggcag 240caaggtcctg gtggccaggg tccctacggg ccgggggcga gtgcggcagc agccgctgca 300ggcggttatg gtccaggaag cggacagcaa ggtccgggag gtcaaggtcc gtatggccca 360ggctctagcg cggctgccgc tgccgcgggt ggcaacggac cagggagcgg acaacagggc 420gcgggacaac agggtccagg acagcaaggc ccaggggcgt cggcggctgc agcggcggcc 480ggaggctatg gacccggctc aggacaacag ggaccgggtc aacaaggacc cggtggccaa 540ggcccctatg gcccgggcgc cagcgcggcc gcagccgccg cgggcgggta cggccccggt 600agcggccagg gaccaggtca gcaggggcca ggaggtcagg gcccatacgg tccgggcgca 660tccgcggcgg cggcagcggc aggtggctac ggtcccggaa gcggccaaca ggggccaggg 720caacaaggac caggacaaca aggtcctggg ggccaaggac cgtatggacc aggagcatca 780gctgcagccg cggcagctgg cggttacggt ccaggctacg gccagcaggg tccgggtcag 840cagggaccgg gaggccaggg gccttatggc cctggcgctt ccgcagccag tgccgcttct 900ggaggatacg ggccgggaag cggtcagcaa ggccctggcc aacaaggacc tggaggccaa 960gggccctacg gcccaggagc ctcggcagcc gcagctgccg caggtgggta tgggccaggt 1020agcgggcaac aagggccggg tcagcaagga ccggggcaac agggacctgg gcagcaagga 1080cccgggggtc aaggcccgta cggacctggt gcgtctgcag ctgctgctgc ggctggtgga 1140tatggtccgg gatcggggca gcagggtccc ggtcagcagg gccctggtca gcaagggcca 1200ggccaacagg gacccggaca acaaggcccg ggtcaacagg gtcctggaca gcaggggccg 1260ggccaacaag gccctgggca acagggtccg gggggacagg gggcctatgg gcctggcgca 1320tctgccgccg ctggcgcagc cggtgggtac gggcctgggt caggtcaaca ggggcctggt 1380caacaaggcc ccgggcaaca gggccccggc cagcaaggtc cagggcagca gggcccggga 1440cagcaagggc ctggacaaca ggggcccgga cagcagggac cttacgggcc cggtgcgagc 1500gcagcggccg ccgccgcagg gggatatggc cccggatcgg gccagcaggg accaggccag 1560caaggacctg gccaacaggg cccggggggt caggggccgt atggtcccgg cgctgcaagt 1620gctgcagtgt ccgtttctag agcacgagcc ggttcgggac aacaagggcc tggccagcag 1680ggcccaggtc aacaagggcc aggacagcag ggtccttatg ggcccggcgc aagcgcagca 1740gctgcggccg ctggtggcta tggtcctggc tccggtcaac agggcccttc gcaacaaggt 1800cccgggcagc aaggtcctgg tggccagggt ccctacgggc cgggggcgag tgcggcagca 1860gccgctgcag gcggttatgg tccaggaagc ggacagcaag gtccgggagg tcaaggtccg 1920tatggcccag gctctagcgc ggctgccgct gccgcgggtg gcaacggacc agggagcgga 1980caacagggcg cgggacaaca gggtccagga cagcaaggcc caggggcgtc ggcggctgca 2040gcggcggccg gaggctatgg acccggctca ggacaacagg gaccgggtca acaaggaccc 2100ggtggccaag gcccctatgg cccgggcgcc agcgcggccg cagccgccgc gggcgggtac 2160ggccccggta gcggccaggg accaggtcag caggggccag gaggtcaggg cccatacggt 2220ccgggcgcat ccgcggcggc ggcagcggca ggtggctacg gtcccggaag cggccaacag 2280gggccagggc aacaaggacc aggacaacaa ggtcctgggg gccaaggacc gtatggacca 2340ggagcatcag ctgcagccgc ggcagctggc ggttacggtc caggctacgg ccagcagggt 2400ccgggtcagc agggaccggg aggccagggg ccttatggcc ctggcgcttc cgcagccagt 2460gccgcttctg gaggatacgg gccgggaagc ggtcagcaag gccctggcca acaaggacct 2520ggaggccaag ggccctacgg cccaggagcc tcggcagccg cagctgccgc aggtgggtat 2580gggccaggta gcgggcaaca agggccgggt cagcaaggac cggggcaaca gggacctggg 2640cagcaaggac ccgggggtca aggcccgtac ggacctggtg cgtctgcagc tgctgctgcg 2700gctggtggat atggtccggg atcggggcag cagggtcccg gtcagcaggg ccctggtcag 2760caagggccag gccaacaggg acccggacaa caaggcccgg gtcaacaggg tcctggacag 2820caggggccgg gccaacaagg ccctgggcaa cagggtccgg ggggacaggg ggcctatggg 2880cctggcgcat ctgccgccgc tggcgcagcc ggtgggtacg ggcctgggtc aggtcaacag 2940gggcctggtc aacaaggccc cgggcaacag ggccccggcc agcaaggtcc agggcagcag 3000ggcccgggac agcaagggcc tggacaacag gggcccggac agcagggacc ttacgggccc 3060ggtgcgagcg cagcggccgc cgccgcaggg ggatatggcc ccggatcggg ccagcaggga 3120ccaggccagc aaggacctgg ccaacagggc ccggggggtc aggggccgta tggtcccggc 3180gctgcaagtg ctgcagtgtc cgttggaggt tacggccctc agtcttcgtc tgttccggtg 3240gcgtccgcag ttgcgagtag actgtcttca cctgctgctt catcgcgagt atcgagcgct 3300gtttcgtctc ttgtctcgtc gggtcccacg aaacatgccg ccctttcaaa tacgatttca 3360tctgtagtgt cccaagttag tgcaagtaac ccggggttat ccggatgcga cgttctcgtt 3420caggcactcc tagaagtagt atccgcgttg gtgagcatct taggcagctc ctcgataggt 3480caaataaact atggtgcttc agcccagtat acacagatgg tgggacagag cgtcgcgcag 3540gcattggctt aa 355273465DNAArtificialADF3Kai-Large-NRSH1 7atgcatcacc atcatcatca tcaccaccac cattcctcgg gctcatcctt ggaagtgtta 60tttcaaggac cagcacgagc cggttcggga caacaagggc ctggccagca gggcccaggt 120caacaagggc caggacagca gggtccttat gggcccggcg caagcgcagc agctgcggcc 180gctggtggct atggtcctgg ctccggtcaa cagggccctt cgcaacaagg tcccgggcag 240caaggtcctg gtggccaggg tccctacggg ccgggggcga gtgcggcagc agccgctgca 300ggcggttatg gtccaggaag cggacagcaa ggtccgggag gtcaaggtcc gtatggccca 360ggctctagcg cggctgccgc tgccgcgggt ggcaacggac cagggagcgg acaacagggc 420gcgggacaac agggtccagg acagcaaggc ccaggggcgt cggcggctgc agcggcggcc 480ggaggctatg gacccggctc aggacaacag ggaccgggtc aacaaggacc cggtggccaa 540ggcccctatg gcccgggcgc cagcgcggcc gcagccgccg cgggcgggta cggccccggt 600agcggccagg gaccaggtca gcaggggcca ggaggtcagg gcccatacgg tccgggcgca 660tccgcggcgg cggcagcggc aggtggctac ggtcccggaa gcggccaaca ggggccaggg 720caacaaggac caggacaaca aggtcctggg ggccaaggac cgtatggacc aggagcatca 780gctgcagccg cggcagctgg cggttacggt ccaggctacg gccagcaggg tccgggtcag 840cagggaccgg gaggccaggg gccttatggc cctggcgctt ccgcagccag tgccgcttct 900ggaggatacg ggccgggaag cggtcagcaa ggccctggcc aacaaggacc tggaggccaa 960gggccctacg gcccaggagc ctcggcagcc gcagctgccg caggtgggta tgggccaggt 1020agcgggcaac aagggccggg tcagcaagga ccggggcaac agggacctgg gcagcaagga 1080cccgggggtc aaggcccgta cggacctggt gcgtctgcag ctgctgctgc ggctggtgga 1140tatggtccgg gatcggggca gcagggtccc ggtcagcagg gccctggtca gcaagggcca 1200ggccaacagg gacccggaca acaaggcccg ggtcaacagg gtcctggaca gcaggggccg 1260ggccaacaag gccctgggca acagggtccg gggggacagg gggcctatgg gcctggcgca 1320tctgccgccg ctggcgcagc cggtgggtac gggcctgggt caggtcaaca ggggcctggt 1380caacaaggcc ccgggcaaca gggccccggc cagcaaggtc cagggcagca gggcccggga 1440cagcaagggc ctggacaaca ggggcccgga cagcagggac cttacgggcc cggtgcgagc 1500gcagcggccg ccgccgcagg gggatatggc cccggatcgg gccagcaggg accaggccag 1560caaggacctg gccaacaggg cccggggggt caggggccgt atggtcccgg cgctgcaagt 1620gctgcagtgt ccgtttctag agcacgagcc ggttcgggac aacaagggcc tggccagcag 1680ggcccaggtc aacaagggcc aggacagcag ggtccttatg ggcccggcgc aagcgcagca 1740gctgcggccg ctggtggcta tggtcctggc tccggtcaac agggcccttc gcaacaaggt 1800cccgggcagc aaggtcctgg tggccagggt ccctacgggc cgggggcgag tgcggcagca 1860gccgctgcag gcggttatgg tccaggaagc ggacagcaag gtccgggagg tcaaggtccg 1920tatggcccag gctctagcgc ggctgccgct gccgcgggtg gcaacggacc agggagcgga 1980caacagggcg cgggacaaca gggtccagga cagcaaggcc caggggcgtc ggcggctgca 2040gcggcggccg gaggctatgg acccggctca ggacaacagg gaccgggtca acaaggaccc 2100ggtggccaag gcccctatgg cccgggcgcc agcgcggccg cagccgccgc gggcgggtac 2160ggccccggta gcggccaggg accaggtcag caggggccag gaggtcaggg cccatacggt 2220ccgggcgcat ccgcggcggc ggcagcggca ggtggctacg gtcccggaag cggccaacag 2280gggccagggc aacaaggacc aggacaacaa ggtcctgggg gccaaggacc gtatggacca 2340ggagcatcag ctgcagccgc ggcagctggc ggttacggtc caggctacgg ccagcagggt 2400ccgggtcagc agggaccggg aggccagggg ccttatggcc ctggcgcttc cgcagccagt 2460gccgcttctg gaggatacgg gccgggaagc ggtcagcaag gccctggcca acaaggacct 2520ggaggccaag ggccctacgg cccaggagcc tcggcagccg cagctgccgc aggtgggtat 2580gggccaggta gcgggcaaca agggccgggt cagcaaggac cggggcaaca gggacctggg 2640cagcaaggac ccgggggtca aggcccgtac ggacctggtg cgtctgcagc tgctgctgcg 2700gctggtggat atggtccggg atcggggcag cagggtcccg gtcagcaggg ccctggtcag 2760caagggccag gccaacaggg acccggacaa caaggcccgg gtcaacaggg tcctggacag 2820caggggccgg gccaacaagg ccctgggcaa cagggtccgg ggggacaggg ggcctatggg 2880cctggcgcat ctgccgccgc tggcgcagcc ggtgggtacg ggcctgggtc aggtcaacag 2940gggcctggtc aacaaggccc cgggcaacag ggccccggcc agcaaggtcc agggcagcag 3000ggcccgggac agcaagggcc tggacaacag gggcccggac agcagggacc ttacgggccc 3060ggtgcgagcg cagcggccgc cgccgcaggg ggatatggcc ccggatcggg ccagcaggga 3120ccaggccagc aaggacctgg ccaacagggc ccggggggtc aggggccgta tggtcccggc 3180gctgcaagtg ctgcagtgtc cgttggaggt tacggccctc agtcttcgtc tgttccggtg 3240gcgtccgcag ttgcgagtag actgtcttca cctgctgctt catcgcgagt atcgagcgct 3300gtttcgtctc ttgtctcgtc gggtcccacg aaacatgccg ccctttcaaa tacgatttca 3360tctgtagtgt cccaagttag tgcaagtaac ccggggttat ccggatgcga cgttctcgtt 3420caggcactcc tagaagtagt atccgcgttg gtgagcatct tataa 3465820DNAArtificialT7 promoter primer 8taatacgact cactataggg 20927DNAArtificialRep Xba I primer 9tctagaaacg gacactgcag cacttgc 271028DNAArtificialXba I Rep primer 10tctagagcac gagccggttc gggacaac 281119DNAArtificialT7 terminator primer 11gctagttatt gctcagcgg 19125PRTArtificialREP6-U1UNSURE(1)..(5)Xaa at position 4 or 5 is any one of amino acids, in particular, it preferably is Ala, Ser, Tyr, Gln, Val, Leu or Ile, it more preferably is Ala, Ser, Tyr, Gln or Val. 12Gly Pro Gly Xaa Xaa 1 5 135PRTArtificialREP6-U2UNSURE(1)..(5)Xaa at position 5 is any one of amino acids, in particular, it preferably is Ala, Ser, Tyr, Gln, Val, Leu or Ile, it more preferably is Ala, Ser, Tyr, Gln or Val. 13Gly Pro Gly Gly Xaa 1 5

Claims

1. A polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent, wherein the solution has a moisture content of less than 5% by mass based on 100% by mass of the solution.

2. The polar solvent solution according to claim 1, wherein the polyamino acid is a polypeptide.

3. The polar solvent solution according to claim 1, wherein the polar solvent contains at least one selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP).

4. The polar solvent solution according to claim 2, wherein the polypeptide is a structural protein.

5. The polar solvent solution according to claim 4, wherein the structural protein includes a crystal region.

6. The polar solvent solution according to claim 2, wherein the polypeptide is a spider silk protein.

7. A method for producing a polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent, comprising: changing a moisture content of the solution to adjust a viscosity of the solution.

8. A method for producing a polar solvent solution in which a solute containing a polyamino acid is dissolved in a polar solvent, comprising: reducing a moisture content of the solution to increase a viscosity of the solution.

9. The method for producing a polar solvent solution according to claim 7, wherein the polyamino acid is a polypeptide.

10. The method for producing a polar solvent solution according to claim 7, wherein the solution has a moisture content of less than 5% by mass based on 100% by mass of the solution.

11. The method for producing a polar solvent solution according to claim 7, wherein the polar solvent contains at least one selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP).

12-14. (canceled)

15. The method for producing a polar solvent solution according to claim 7, wherein the moisture content of the solution is changed by drying the solute containing the polyamino acid and thereafter dissolving it in the polar solvent.

16. The method for producing a polar solvent solution according to claim 7, wherein the moisture content of the solution is changed by adjusting a relative humidity of an atmosphere in the production and/or storage of the solution.

17. The method for producing a polar solvent solution according to claim 16, wherein the relative humidity of the atmosphere in the production and/or storage of the solution is adjusted to 1.3% RH or less.

18. The method for producing a polar solvent solution according to claim 8, wherein the polyamino acid is a polypeptide.

19. The method for producing a polar solvent solution according to claim 8, wherein the solution has a moisture content of less than 5% by mass based on 100% by mass of the solution.

20. The method for producing a polar solvent solution according to claim 8, wherein the polar solvent contains at least one selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP).

21. The method for producing a polar solvent solution according to claim 8, wherein the moisture content of the solution is changed by drying the solute containing the polyamino acid and thereafter dissolving it in the polar solvent.

22. The method for producing a polar solvent solution according to claim 8, wherein the moisture content of the solution is changed by adjusting a relative humidity of an atmosphere in the production and/or storage of the solution.

23. The method for producing a polar solvent solution according to claim 22, wherein the relative humidity of the atmosphere in the production and/or storage of the solution is adjusted to 1.3% RH or less.