KR101841482B1

KR101841482B1 - A human fibroblast growth factor-9 mutant with high stability and use of the same

Info

Publication number: KR101841482B1
Application number: KR1020160022666A
Authority: KR
Inventors: 신항철; 박연희; 오종광; 김동환; 김수진; 박예영
Original assignee: (주)피앤피바이오팜
Priority date: 2016-02-25
Filing date: 2016-02-25
Publication date: 2018-03-23
Also published as: KR20170100288A

Abstract

The present invention relates to a high-stability fibroblast growth factor-9 mutant and its use, and more particularly, to a high-stability fibroblast growth factor-9 mutant and its use. More specifically, A high-stability fibroblast growth factor-9 (FGF-9) mutant, a DNA base sequence encoding the FGF-9 variant, an expression vector comprising the DNA base sequence, , A method for producing the FGF-9 mutant, and a composition comprising the FGF-9 variant as an active ingredient. According to the present invention, the FGF-9 mutant of the present invention is excellent in heat stability and stability in an aqueous solution state, so that functional cosmetics which do not lose activity unlike conventional natural FGF-9 products, Wound cloning and hair growth, hair growth, and hair loss treatment.

Description

A human fibroblast growth factor-9 mutant having increased stability and a use thereof include a human fibroblast growth factor-9 mutant with high stability and use of the same,

The present invention relates to high-stability human fibroblast growth factor-9 variants and uses thereof.

Growth factors play an important role in regulating cell growth, proliferation, and differentiation. Therefore, there is a system that naturally repairs the damage and aging of the skin caused by internal and external factors such as wound, surgery, etc., and the growth factor plays an important role here. In order to maintain the function of each tissue, various growth factors are generated and maintained at a constant concentration. As the age increases, the concentration of growth factors decreases in each tissue such as the skin, and aging progresses as the cell regeneration and division function is weakened to form wrinkles and decrease elasticity.

Among them, FGF-9 (Fibroblast Growth Factor-9, FGF-9) consists of 205 amino acids and is composed of a polypeptide having a molecular weight of 23.14 Dalton. It plays an important role in promoting the generation and growth of hair through development, angiogenesis, wound healing, new hair follicle cell regeneration, hair follicle cell regeneration and differentiation, inhibiting androgenic hair loss, and tissue regeneration. FGF-9 produced in gamma-delta-T cells in the dermis promotes intracellular WNT-signal production, promotes cell regeneration, and promotes regeneration and growth differentiation of hair follicle cells.

However, in the case of growth factors present in these blood and tissues, its half-life is known to be very short as several minutes.

In addition, the bioavailability of protein therapeutics such as FGF-9 is often limited by short plasma half-lives and susceptibility to proteases, hindering maximum clinical efficacy. In order to use and develop FGF-9 more effectively, physico-chemical stability in vitro as well as stability in the body should be improved so that the use of FGF-9 in the manufacture, storage and distribution of quasi-drugs and creams will increase.

Therefore, there is a need to develop new FGF-9 variants that are more stable and active.

The present inventors have made an effort to develop a high-stability fibroblast growth factor-9 mutant. More specifically, in order to increase the stability of the FGF-9 protein, a molecular design method in which the amino acid sequence of a protein is modified is applied to obtain a candidate group with improved thermal stability. And confirming excellent effects, thereby completing the present invention.

Therefore, an object of the present invention is to provide a high-stability fibroblast growth factor-9 variant.

Another object of the present invention is to provide a gene encoding a FGF-9 mutant.

Another object of the present invention is to provide a cosmetic composition for preventing hair loss or improving skin condition.

It is still another object of the present invention to provide a pharmaceutical composition for preventing hair loss or preventing or treating skin diseases.

In order to achieve the above object, the present invention provides a high-stability fibroblast growth factor variant in which at least one amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with cysteine, and one amino acid on the surface thereof is substituted with another amino acid.

The term "fibroblast growth factor-9" or "FGF-9 ", as used herein, refers to a basic protein (pI 7.2) with a molecular weight of about 23 kDa that is secreted predominantly in gamma-delta- It is known to promote the growth of mesenchymal stem cells. In addition, it is known that the secretion of FGF-9 is reduced in adults and the formation of new hair follicle cells is decreased. As a result, it is known that the amount of FGF-9 secreted in human hair follicle cells is small. In addition, it is a protein that promotes the growth of endothelial cells and smooth muscle cells, and exhibits excellent efficacy in trauma treatment and angiogenesis, increases the synthesis of collagen and elastin, maintains skin elasticity, and induces new hair follicle cell growth It is known that hair regeneration and growth through regeneration and differentiation of hair follicle cells inhibits androgenic hair loss. It is also known to help normal cell growth, promote wound healing, and perform its healing function.

The mutant of the present invention can be obtained by selecting a site which is not related to the active site of FGF-9 through a method of homology alignment between species and the tertiary structure of FGF-9 and protein molecular modeling using a computer, , Characterized in that stability is improved by reducing loop entropy by additionally producing a disulfide bond by replacing one residue near the loop in FGF-9 with cysteine. Further, by selecting one residue outside FGF-9 and replacing it with another amino acid, the stability of the protein is increased by increasing the hydrogen bonding and van der Waals bonding, thereby adding water solubility and structure stabilization of the protein.

In one embodiment of the present invention, the mutant is preferably a mutant in which the 63rd and 96th motifs of SEQ ID NO: 1 are substituted with cysteine, more preferably the mutant is a mutant in which the 63rd and 96th motifs of SEQ ID NO: Lt; / RTI >

The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, The amino acid is substituted with mesaionine, the amino acid at position 184 of SEQ ID NO: 1 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

In one embodiment of the present invention, the mutant is preferably a mutant in which the 63rd and 192th positions of SEQ ID NO: 1 are substituted with cysteine, more preferably the 63rd and 192th mutations of SEQ ID NO: 1 are cysteine Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant substituted by the 65th and 190th cysteines in SEQ ID NO: 1, more preferably the mutant is a mutant in which the 65th and 190th mutations in SEQ ID NO: 1 are cysteine Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant substituted with cysteine at positions 66 and 187 of SEQ ID NO: 1, more preferably the mutant at positions 66 and 187 of SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant in which the 187th position of SEQ ID NO: 1 is substituted with cysteine, more preferably the mutant is substituted with cysteine at position 187 of SEQ ID NO: 1,

In one embodiment of the present invention, the mutant is preferably a mutant in which the 166th position of SEQ ID NO: 1 is substituted with cysteine, more preferably the mutant is substituted with cysteine at position 166 of SEQ ID NO: 1,

In one embodiment of the present invention, the mutant is preferably a mutant in which the 69th and 183rd of SEQ ID NO: 1 are substituted with cysteine, and more preferably the 69th and 183th mutants of SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant in which the 72nd and 84th cysteines in SEQ ID NO: 1 are substituted with cysteine, more preferably the mutant is the 72nd and 84th cysteines in SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant in which the 72nd and 170th motifs of SEQ ID NO: 1 are substituted with cysteine, more preferably the 72th and 170th motifs of SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant in which the 75th and 93rd residues of SEQ ID NO: 1 are substituted with cysteine, more preferably the 75th and 93th residues of SEQ ID NO: Lt; / RTI >

In another embodiment of the present invention, the mutant is preferably a mutant in which the 76th and 109th motifs of SEQ ID NO: 1 are substituted with cysteine, more preferably the mutant is a mutant of the 76th and 109th motifs of SEQ ID NO: Cysteine,

The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, The amino acid at position 184 of SEQ ID NO: 1 is replaced by tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is replaced by histidine, but the present invention is not limited thereto.

In one embodiment of the present invention, the mutant is preferably a mutant substituted with cysteine at positions 80 and 126 of SEQ ID NO: 1, more preferably mutant at positions 80 and 126 of SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant in which the 82nd and 124th motifs of SEQ ID NO: 1 are substituted with cysteine, more preferably the mutant is a mutant of cysteine 82 and 124 in SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant substituted with cysteine at the 84th and 170th positions in SEQ ID NO: 1, more preferably the mutant at 84th and 170th positions in SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant in which the 94th and 110th motifs of SEQ ID NO: 1 are substituted with cysteine, more preferably the mutant is a mutant of 94th and 110th motifs of SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant in which the 114th and 126th cysteines in SEQ ID NO: 1 are substituted with cysteine, more preferably the mutant is a mutant in which the 114th and 126th cysteines of SEQ ID NO: Lt; / RTI >

The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, The amino acid is substituted with mesaionine, the amino acid at position 184 of SEQ ID NO: 1 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine. In one embodiment of the present invention, the mutant is preferably a mutant in which the 122nd and 175th of SEQ ID NO: 1 are substituted with cysteine, more preferably the mutant is a mutant of cysteine 122 and 175 of SEQ ID NO: Lt; / RTI >

In one embodiment of the present invention, the mutant is preferably a mutant substituted with cysteine at positions 165 and 178 of SEQ ID NO: 1, more preferably the mutant at positions 165 and 178 of SEQ ID NO: Lt; / RTI >

In another embodiment of the present invention, the mutant is preferably a mutant substituted with the 117th cysteine in SEQ ID NO: 1, more preferably the mutant is a mutant in which the 117th mutation in SEQ ID NO: 1 is substituted with cysteine,

In one embodiment of the present invention, the amino acid substituted with cysteine is selected from the group consisting of 63rd arginine, 65th glutamine, 66th leucine, 69th arginine, 72nd phenylalanine, 75th glutamic acid, 76 The second isosurine, the 80th glycine, the 82th isoisine, the 84th glycine, the 93rd glycine, the 94th isoleucine, the 96th glutamic acid, the 109th glycine, the 110th valine, the 114th leucine, the 117th glycine, 122 The first glycine, the 124th leucine, the 126th glycine, the 165th alanine, the 165th leucine, the 170th glycine, the 175th glycine, the 178th tyrosine, the 183rd lysine, the 187th phenylalanine, the 190th arginine, But it is not limited thereto.

The present invention also provides a gene encoding the mutant of the present invention.

In one embodiment of the present invention, the gene is preferably composed of one DNA base sequence selected from the group consisting of SEQ ID NOS: 2 to 30, but mutations such as one or more substitutions, deletions, inversions, All mutants that achieve the desired effect of the invention are also included within the scope of the present invention.

The FGF-9 mutant of the present invention increases the stability against heat while maintaining the protein activity as compared with the wild type. As shown in the following Experimental Example, it can be seen that the FGF-9 mutant has the activity equivalent to the wild type and the stability against heat is also remarkably increased.

The present invention also provides an expression vector comprising the gene.

The expression vector of the present invention can be prepared by inserting the gene of FGF-9 into a general expression vector. In the preferred embodiment of the present invention, the pET21a vector is used as an expression vector, but not always limited thereto, and any cell expression vector generally used can be used. In a preferred embodiment of the present invention, a vector having the FGF-9 gene inserted into the pET21a vector was prepared and named "pSSB-FGF-9" (FIG.

The present invention also provides a transformant transformed with said expression vector.

The present invention also provides a cosmetic composition for improving skin condition comprising the above-described high-stability FGF-9 mutant of the present invention as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating skin diseases comprising the above-described high-stability FGF-9 variant of the present invention as an active ingredient.

The present invention also provides a cosmetic composition for improving hair growth and hair loss comprising the above-described high-stability FGF-9 variant of the present invention as an active ingredient.

The FGF-9 mutant of the present invention can be produced by a method of expressing an FGF-9 mutant by transforming a host cell with a vector containing a gene encoding a FGF-9 mutant produced by a site-specific mutagenesis method or the like, Can be produced by a chemical amino acid synthesis method.

The DNA encoding the FGF-9 mutant is a DNA encoding the amino acid of the substitution site of the native FGF-9.

On the other hand, it is well known that the nucleotide sequence of a DNA encoding the same amino acid sequence may differ due to the presence of a large number of codons encoding one amino acid by degeneracy of the codon.

DNA coding for the FGF-9 mutant can be chemically synthesized, or can be prepared by preparing a wild-type FGF-9 cDNA and using site-directed mutagenesis based thereon.

The prepared DNA encoding the FGF-9 variant of the present invention can be expressed using any suitable prokaryotic or eukaryotic expression system well known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual , 2nd ed., Cold Spring Habor Laboratory, Cold Spring Habor Laboratory Press, USA, 1989).

Expression is preferably performed in Escherichia coli such as Escherichia coli BL21 (DE3), Escherichia coli JM109 (DE3), Escherichia coli NM522 and the like in the case of ungalactic FGF-9 variants, and suitable vectors that can be used for expression in E. coli (Proc. 8th Int. Biotechnology Symposium, Soc. Frac, de Microbiol., Paris, (Durand et al., Eds.), Pp. 680 -697, 1988).

Transformation of the host cell by the vectors described above can be performed by any of the conventional methods (Sambrooketal., Molecular Cloning, Alaboratory Manual, 1989; Ito et al., J. Bacteriol. 153: 263, 1983) .

When E. coli is transformed, a competent cell capable of absorbing DNA can be prepared, followed by treatment according to a known method or the like.

According to another aspect of the present invention, the present invention provides a method of producing a high-stability basic fibroblast growth factor (FGF-9) mutant comprising the steps of: (a) ; &Lt; / RTI > And (b) separating the mutant from the culture obtained in the step (a).

According to a preferred embodiment of the present invention, step (b) of the method comprises the steps of: (c) disrupting the transformant and separating the aggregate, (d) removing the separated aggregate, (e) Separating and purifying the aggregate-removed supernatant using ion exchange resin chromatography; And (f) separating and purifying the high-stability basic fibroblast growth factor variant after the ion exchange resin using heparin affinity chromatography.

In general, host microorganisms containing the objective expression vector are cultured under their optimal growth conditions to the extent that they maximize production of the desired protein. For example, Escherichia coli BL21 (DE3) cells transformed with a vector containing the ampicillin resistance gene as a selection marker are cultured at 37 DEG C in LB medium containing ampicillin.

Recovery and purification of the produced FGF-9 variants after culturing the transformed host cells can be carried out by various methods known in the art or by using them in combination. For example, FGF-9 variants expressed in transformed E. coli cells can be recovered from the cell culture or after extraction of the cells by a suitable method known to the proteochemical system.

Preferably, in order to purify the FGF-9 variant, the culture medium of the recombinant E. coli cells is centrifuged to harvest the cells, and the harvested cells are suspended in a buffer solution to which lysozyme is added and disrupted by ultrasonication. The cell lysate is centrifuged to separate the insoluble granular aggregates, and the separated aggregates are removed. The supernatant having the above aggregates removed is separated and purified using ion exchange resin chromatography, and the ion-exchange resin is then separated and purified using heparin affinity chromatography to obtain the resultant high-stability FGF-9 variant.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating skin diseases comprising the high-stability FGF-9 variant as an active ingredient.

As demonstrated in the following examples, the high-stability FGF-9 variants of the present invention have the same activity as native FGF-9, have excellent thermal stability and stability in aqueous solution. Therefore, the composition of the present invention is very effective for preventing or treating skin diseases.

Preferably, the composition of the present invention is used for the treatment of skin such as skin inflammation, acute chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simplex poisoning, biliary cysts, deprived dermatitis, bulimia nervosa, psoriasis, It is used for the prevention or treatment of diseases.

In addition, the composition of the present invention can provide a composition for treating wound.

Preferably, the composition of the present invention is used for the treatment of closed wounds and open wounds. Examples of closed windows include contusion or burys and examples of open windows include abrasion, laceration, avulsion, penetrated wound, and gun shot wound.

In addition, the composition of the present invention can be used for treatment for hair growth and hair loss treatment.

The composition of the present invention comprises (a) a pharmaceutically effective amount of the aforementioned FGF-9 variant of the present invention; And (b) a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve efficacy or activity of the FGF-9 variant described above.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, trehalose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, But is not limited thereto. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, local administration, .

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the preferred daily dose of the pharmaceutical composition of the present invention is 0.0001-10 mg / kg body weight.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets, capsules or gels (e.g., hydrogels), and may additionally contain dispersing or stabilizing agents .

According to another aspect of the present invention, there is provided a cosmetic composition for skin condition improvement comprising the above-mentioned high-stability FGF-9 variant as an active ingredient.

According to still another aspect of the present invention, there is provided a hair loss improving cosmetic composition and a hair growth inducing cosmetic composition comprising the high-stability FGF-9 variant as an active ingredient.

As demonstrated in the following examples, the high-stability FGF-9 variants of the present invention have the same activity as native FGF-9, have excellent thermal stability and stability in aqueous solution. Therefore, the composition of the present invention is very effective for improving the skin condition.

Preferably, the composition of the present invention is used for the improvement of skin conditions such as wrinkle improvement, skin elasticity improvement, skin aging prevention, hair loss prevention or promotion of hair growth, skin moisturization improvement, black spot removal or acne treatment.

The composition of the present invention comprises (a) a cosmetically effective amount of the above-mentioned FGF-9 variant of the present invention; And (b) an cosmetically acceptable carrier.

The term "cosmetically effective amount" as used herein means an amount sufficient to achieve the skin-improving effect of the composition of the present invention described above.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, Oils, powder foundations, emulsion foundations, wax foundations and sprays, but are not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The components contained in the cosmetic composition of the present invention include, in addition to the FGF-9 variant and the carrier component as the active ingredient, components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, And may contain the same conventional adjuvants.

Since the compositions of the present invention include the above-described high-stability FGF-9 mutant of the present invention as an active ingredient, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

According to the present invention, the FGF-9 mutant of the present invention is excellent in heat stability and stability in an aqueous solution state, so that production of a functional cosmetic product which does not lose activity unlike the existing natural FGF-9 product during distribution and storage, It can be used for cloning and balding hair growth hair growth and so on.

Figure 1 shows an overview of the assembly of the plasmid pSSB-FGF-9.
FIG. 2 is a chart comparing cell activity of wild type and mutants in the BALB / 3T3 cell line. FIG.
FIG. 3 is a graph comparing cell activities after incubation at 25 ° C for 28 days to confirm the stability of the native form and mutants.
FIG. 4 is a graph comparing cell activity after incubation at 50 ° C. for 7 days to confirm the stability of the native form and mutants.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Experimental Methods and Materials

DNA construction

The protein expression vector pET21a (Fig. 1A) and E. coli expressing strain BL21 (DE3) and Rosetta (DE3) were purchased from Novagen and Top10 was used for the E. coli strain for cloning. All of the restriction enzymes used in the recombination were NEB (New England Biolabs) and the ligase was Roche T4 DNA ligase. Ex Taq DNA polymerase used in PCR is a product of Takara, and pfuUltra ^{™ H} F DNA polymerase used in point mutation is a product of Agilent. The DNA gel extraction kit and the plasmid mini prep kit are products of Cosmojin Tech. In addition, primers were prepared by Cosmojin Tech Co., Ltd., and DNA sequencing was performed by Cosmojin Tech Co., Ltd.

Protein expression

The expression derivatives IPTG (isopropyl-1-thio-beta-D-galactopyranoside) and ampicillin and chloramphenicol used as antibiotics were purchased from Sigma. E. coli Bacto tryptone, yeast extract, which was used to make LB medium, was purchased from BD (Becton Dicknson), and NaCl was purchased from Duksan.

Protein purification

The reagents used in the purification process are as high in purity as possible, and the reagents used in the purification process are as follows. Sodium phosphate monobasic (Sigma), sodium phosphate dibasic (Sigma), and sodium chloride (Sigma). Columns used in FPLC were SP-sephrose and heparin affinity columns.

FPLC

FPLC used GE UPC-800.

CD (Circular dichroism)

The CD was a J-810 spectropolarimeter from Jasco.

Homology modeling

Homology modeling was done by Modeller (Andrej Sali).

Energy minimization

Energy minimization was performed using Amber 99FF force field included in Chimera.

Disulfide bond Disulfide predict

YASARA Web server was used to predict the disulfide bond formation.

Disulfide bond distance measurement

Protein contact map visualization (Andreas Viklund.) Was used to measure the disposable distance.

Vector system

A pET21a vector (Novagen) was used as an expression vector to produce mutant FGF-9. The wild-type FGF-9 gene was obtained from Bioneer Co., Ltd. and amplified by PCR (polymerase chain reaction) using the following primers. The PCR product thus obtained was treated with Nde I and Xho I restriction enzymes and inserted into pET21a vector.

Point mutation

In order to increase the stability of FGF-9, the amino acid part to be changed was found through the structure of the protein and the molecular modeling method, and amplified using pfu Ultra ™ DNA polymerase using the following primers using the Quickchange mutagenesis method. The DpnI reaction was performed to remove the natural form of FGF-9 template, and the resultant was transformed into Top 10, and the mutant was confirmed by sequencing.

Natural type And Mutant FGF -9 expression and purification

FGF-9-inserted recombinant vector was transformed into E. coli BL21 (DE3) by heat shock method. The E. coli strain was inoculated in 500 ml LB medium containing 50 ug / ml ampicillin and grown at 37 ° C until the OD ₆₀₀ value reached 0.6. Then, 0.5 mM IPTG (isopropyl-1-thio-beta-D-galactopyranoside) was added and incubated for 4 hours. When the OD ₆₀₀ value was 2.0 or more, cells were centrifuged at 8000 rpm for 10 minutes. The supernatant obtained after cell disruption was purified by a heparin column.

molecule modelling (Molecular modeling)

A candidate group capable of disulfide bonding was set up using 1g82, which is a protein structure registered in the PDB. Protein contact map visualization program was used to analyze the residues with C-alpha carbon distance of less than 6 Å and C-beta carbon distance of 5 Å in two amino acids using a plot. The Yasara energy minimization server was used to analyze the formation of disulfide bonds and energy minimization was performed using the AMBER force field FF99 of the chimera. The structure of the resulting protein was aligned with the native FGF-9 structure, and the structure having the RMSD value of 0.5 or less was found and tested.

Based on the results of the above experiments, the results of the molecular design are attached to Table 1 below.

No Cys mutant location Disulfide bond prediction energy One Natural type (1g82) OK -88484 2 63 96 OK -88831 3 63 192 OK -88509 4 65 190 NO -90580 5 66 187 OK -89181 6 68 187 NO -88984 7 68 166 OK -89384 8 69 183 OK -88198 9 72 84 OK -89026 10 72 170 OK -89310 11 75 93 OK -90548 12 76 109 OK -90716 13 80 126 OK -90433 14 82 124 OK -89803 15 84 170 NO -88621 16 94 110 OK -89734 17 114 126 NO -89933 18 122 175 NO -90662 19 134 117 OK -92183 20 165 178 Ok -89576

For additional solubility and stability, additional mutation sites were selected through molecular design methods to fill the surface residue and FGF-9 internal cavity. The specific position and the calculated energy value are shown in Table 2.

No Cys mutant location energy 21 F184Y -91394 22 F77Y / F92Y -90944 23 V135T -89776 24 L188H -90281 25 L116M -89973 26 L116F -90134 27 v164F -90722 28 v164M -90105 29 Y148F -90421

CD (Circular Dichroism)

FGF-9 was dissolved in 20 mM sodium phosphate (pH 7.0) to make a final concentration of 0.2 mg / ml for the structural analysis and Tm measurement of wild-type FGF-9 and mutants. The sample was loaded into a 0.1 cm cell and the structure was constructed with 190 nm to 250 nm band width 1 nm, response 0.25 sec, data pitch 0.1 nm, scanning speed 20 nm / min, pathlength 1 cm, accumulation 8, Respectively. The melting temperature was 0.2 mg / ml in a 0.1 cm cuvette at 205 nm wavelength at 20 ℃ and 95 ℃. Conditions were measured at 20 ° C to 95 ° C at 1 ° C / min.

Cell proliferation assay

Experiments were carried out using the cell proliferation ability to confirm that the produced wild type FGF-9 and the mutant actually show activity. BALB-3T3 cells were maintained in DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. BALB-3T3 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well culture plate. BALB-3T3 cells cultured for 24 hours were starvated with serum-free DMEM medium and then treated with sample solution in DMEM medium containing 0.5% FBS for each concentration and cultured for 72 hours. After incubation, 10 μl of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide] solution was added and reacted for 2 hours. &Lt; / RTI > Absorbance was measured at 540 nm using a spectrophotometer. Sensitivity to the drug was compared by the percentage of absorbance of the untreated well (control) in the drug treated wells.

Incubation test

Incubation test of wild-type FGF-9 and mutants was performed to confirm the storage at room temperature. Each wild-type FGF-9 and mutants were dissolved at 0.5 mg / ml in 1X PBS (pH 7.8) and incubated in a water bath at 37 ° C and 50 ° C. 1 or 7 days, and centrifuged at 13000 rpm for 15 minutes at 4 ° C to obtain the supernatant liquid, which was then quantitated and analyzed by HPLC.

Example 1 Construction of pSSB-FGF-9 Plasmid Containing Human FGF-9 cDNA < RTI ID = 0.0 >

A DNA encoding FGF-9 was prepared by polymerase chain reaction using a human mononuclear cell cDNA library as a template and a primer. The base sequences of the primers used are as follows:

Sense primer 5'-GGCGGGCATATGCTAGGGGAGGTAGGG-3 '

Antisense primer 3'-CGACTGGAGAGAATGCTCTTAGCAGACAT-5 '.

The FGF-9 portion of FIG. 1B was amplified using the primers described above. One μg of the amplified DNA fragment was dissolved in 50 μl of TE (pH 8.0) solution, and then 2 units of NdeI (NEB) and 2 units of BamHI (NEB), and reacted at 37 ° C for 2 hours to have a NdeI restriction enzyme site at the 5'-end and a BamHI restriction enzyme site at the 3'-end. 20 ng of this DNA fragment was treated with NdeI and BamHI in the same manner, and 20 ng of pET21a (+) plasmid (Novagen) was added to each well of a DNA purification kit (GeneAll) TE (pH 8.0) solution, and then 1 unit of T4 DNA ligase (NEB) was added and allowed to react at 16 ° C for 4 hours. The resulting plasmid was named pSSB-FGF-9 (Fig. 1).

The thus prepared expression plasmid pSSB-FGF-9 was transformed into E. coli BL21 (DE3) by heat shock. Colonies resistant to ampicillin generated in the solid medium after transformation were selected and inoculated into 10 ml of LB / ampicillin. The selected expression strains were incubated at 37 ° C for 12 hours and then mixed with 1: 1 with 100% glycerol and stored at -70 ° C.

The expression strain was inoculated into 10 ml of LB medium (LB / ampicillin) and cultured for more than 12 hours. The LB medium was added and then transferred to the absorbance _{(LB / ampicillin) OD 600 0.4} ~ 0.5 for a final concentration of IPTG (isopropyl-1-thio- beta -D-galactopyranoside) when 500 ml of the so that the 0.5 mM. After incubation at 37 ° C for 4 hours with shaking at 200 rpm, E. coli pellets were obtained by centrifugation at 8000 rpm for 10 minutes. Cells were disrupted by sonication. The column was then purified using FPLC. Fractions containing FGF-9 were identified by SDS-PAGE for each fraction and then quantified by Bradford assay. As a result, 10 mg of FGF-9 was obtained.

&Lt; Example 2: Construction of pSSB-FGF-9 mutant plasmid >

Fifteen pSSB-FGF-9 plasmids were used as a template and pfuUltra F DNA polymerase was used to amplify the 19 pSSB-FGF-9 Mutant plasmids. The plasmid pSSB-FGF-9, which was a template, was digested with DpnI and transformed into E. coli Top10 by heat shock. Colonies resistant to ampicillin generated in the solid medium after transformation were selected and inoculated into 10 ml of LB / ampicillin. After incubation at 37 ° C for 16 hours, DNA prep was performed and sequencing of the obtained DNA confirmed pSSB-FGF-9 mutant plasmids. The nucleotide sequences of the primers used in the above procedure are as follows:

AAA GGA ATA CTG CGC TGC CGG CAA CTT TAT TGC-3 'and antisense primer 3'-GCA ATA AAG TTG CCG GCA GCG CAG when CGC, the codon of the 63rd arginine, is substituted by TGC, the codon of cysteine TAT TCC TTT -5 ';

ATG CTG CGC CGC CGG TGC CTT TAT TGC CGG ACA-3 'and antisense primer 3'-TGT CCG GCA ATA AAG GCA CCG GCG GCG when the CAA, the codon of the 65th glutamine, is replaced by the TGC which is the codon of cysteine CAG TAT-5 ';

CTG CGC CGC CGG CAA TGC TAT TGC CGG ACA GGT -3 'and antisense primer 3'-ACC TGT CCG GCA ATA GCA TTG CCG GCG at the substitution of the 66th codon of lucine with TGC, the codon of cysteine GCG CAG -5 ';

CGG CAA CTT TAT TGC TGC ACA GGT TTT CAT TTG -3 'and the antisense primer 3'-CAA ATG AAA ACC TGT GCA GCA ATA at the substitution of the 69th arginine codon CGG with the codon TGC of the cysteine AAG TTG CCG -5 ';

TTT TGC CGT ACA GGT TGC CAT TTG GAA ATC TTT-3 'and antisense primer 3'-AAA GAT TTC CAA ATG GCA ACC at the time of substitution of the 72 th phenylalanine codon TTT with the codon of cysteine TGC TGT CCG GCA ATA -5 ';

GTC ATT TGT AAA GAT GCA CAT ATG TTT CAT TTG TGC ATC TTT CCA AAT GGC -3 'and antisense primer 3'-GCC ATT TGG AAA GAT CACA ATG AAA ACC TGT -5 ';

When ATC of 76th isoleucine is substituted with TGC which is a codon of cysteine, the sense primer 5'-GGT TTT CAT TTG GAA TGC TTT CCA AAT GGC ACG -3 'and antisense primer 3'-CGT GCC ATT TGG AAA GCA TTC CAA ATG AAA ACC -5 ';

AGT ATC TTT CCA AAT TGC ACG ATA CAG GGG ACT -3 'and antisense primer 3'-AGT CCC CTG TAT CGT GCA ATT TGG AAA GAT TTC -5 ';

ATT, the codon of 82th isoleucine, was substituted with TGC, a codon of cysteine. The sense primer 5'-TTT CCA AAT GGC ACG TGC CAG GGG ACT CGT AAG -3 'and antisense primer 3'-CTT ACG AGT CCC CTG GCA CGT GCC ATT TGG AAA-5 ';

ATG CAT TGC ACT CGT AAG GAT CAT-3 'and antisense primer 3'-ATG ATC CTT ACG AGT GCA CTG TAT CGT at the time of substitution of GGG, codon of 84th glycine, with TGC which is a codon of cysteine GCC ATT-5 ';

CAT TCT CGA TTC GGC TGC CTG GAG TTT ATC TCT -3 'and antisense primer 3'-AGA GAT AAA CTC CAG GCA GCC GAA at the substitution of ATT, the codon of the 94th isoleucine, with TGC, the codon of cysteine TCG AGA ATG-5 ';

GTA GTA GAT AGC GGA TGC TAT CTT GGT ATG AAT -3 'and antisense primer 3'-ATT CAT ACC AAG ATA GCA TCC GCT ATC TAC ACC -5 ';

GGC, a codon of the 122nd glycine, was substituted with TGC, a codon of cysteine. The sense primer 5'-GGT ATG AAT GAA AAA TGC GAA TTA TAC GGA TCA -3 'and antisense primer 3'-TGA TCC GTA TAA TTC GCA TTT TTC ATT CAT ACC -5 ';

CGC CGC TAT TAT GTC TGC CTG AAC AAG GAT GGT-3 'and antisense primer 3'-ACC ATC CTT GTT CAG GCA GAC ATA ATA when substitution of GCG, the codon of 165th alanine, with TGC, the codon of cysteine GCG GCG-5 ';

GAT CAT TCT CGA TTC TGC ATT CTG GAG TTT ATC -3 'and antisense primer 3'-GAT AAA CTC CAG AAT GCA GAA TCG AGA ATG ATC -5 '.

CGA TTC GGC ATT CTG TGC TTT ATC TCT ATT GCT -3 'and antisense primer 3'-AGC AAT AGA GAT AAA GCA CAG AAT GCC GAA TCG -5 '.

TGG AGT ATT CGT TGC GTA GAT AGC GGA TTA -3 'and antisense primer 3'-TAA TCC GCT ATC TAC GCA ACG AAT ACT AAC CAA -5 '.

GTT AGT ATT CGT GGT TGC GAT AGC GGA TTA TAT -3 'and antisense primer 3'-ATA TAA TCC GCT ATC GCA ACC ACG AAT ACT AAC -5 '.

AGC GGA TTA TAT CTT TGC ATG AAT GAA AAA GGC -3 'and antisense primer 3'-GCC TTT TTC ATT CAT GCA AAG ATA TAA at the substitution of codon GGT of 117th glycine with TGC which is a codon of cysteine TCC GCT -5 '.

ATS GAA AAA GGC GAA TGC TAC GGA TCA GAA AAA -3 'and antisense primer 3'-TTT TTC TGA TCC GTA GCA TTC GCC TTT at the substitution of the 124 th lysine codon TGC, which is a codon of cysteine, TTC ATT -5 '.

AAA GGC GAA TTA TAC TGC TCA GAA AAA CTG ACA -3 'and antisense primer 3'-TGT CAG TTT TTC TGA GCA GTA TAA TTC GCC TTT -5 '.

CGT TAT TAT GTC GCG TGC AAC AAG GAT GGT ACG-3 'and antisense primer 3'-CGT ACC ATC CTT GTT GCA CGC GAC ATA at the substitution of the 166th lysine codon CTG with the codon TGC of cysteine ATA GCG -5 '.

GCG CTG AAC AAG GAT TGC ACG CCG AGA GAA GGC -3 'and antisense primer 3'-GCC TTC TCT CGG CGT GCA ATC CTT GTT when the codon GGT of the 170th glycine was substituted with TGC which is the codon of cysteine CAG CGC -5 '.

GGC, the codon of the 175th glycine synthase, is replaced with TGC, a cysteine codon. Sense primer 5'- GGT ACG CCG AGA GAA TGC ACC AGA ACC AAA AGG -3 'and antisense primer 3'- CCT TTT GGT TCT GGT GCA TTC TCT CGG CGT ACC -5 '.

AGA GAA GGC ACC AGA TGC AAA AGG CAT CAA AAA -3 'and antisense primer 3'- TTT TTG ATG CCT TTT GCA TCT GGT GCC TTC TCT-5 '.

ACCA AAA AGG CAT CAA TGC TTT ACT CAC TTC CTG -3 'and antisense primer 3'-CAG GAA GTG AGT AAA GCA TTG ATG CCT when AAA, the codon of 183rd lysine, is substituted by TGC, a codon for cysteine TTT GGT-5 '.

CTA GTT-3 'and an antisense primer 3'-AAC GGG ACG AGG CAG GCA GTG AGT AAA TTT ACT CAC AAA TTT ACT CAC AAG TTT ACT CAC TGC CTG CCT CGT CTA GTT-3' and antisense primer 3'- TTT TTG -5 '.

ACT CAC TTC CTG CCT TGC CCC GTT GAT CCG GAT -3 'and antisense primer 3'-ATC CGG ATC AAC GGG GCA AGG CAG GAA when the CGT, the codon of the 190th arginine, is replaced by the TGC, the codon of cysteine GTG AGT-5 '.

TTC CTG CCT CGT CCC TGC GAT CCG GAT AAG GTT -3 'and antisense primer 3'-AAC CTT ATC CGG ATC GCA GGG ACG AGG at the substitution of GTT, the 192 th Bellin codon, with TGC, the codon of cysteine CAG GAA-5 '.

In order to introduce additional mutations (Table 2) selected through the molecular design method, a second-order mutant plasmid was prepared by PCR using a first mutant plasmid as a template and two complementary primers corresponding to each mutant . The first mutant plasmid of pSSB-FGF-9, which was a template, was digested with DpnI and transformed into E. coli Top10 by heat shock. Colonies resistant to ampicillin generated in the solid medium after transformation were selected and inoculated into 10 ml of LB / ampicillin. After incubation at 37 ° C for 16 hours, the DNA preparation was performed and sequencing of the obtained DNA confirmed the plasmid of pSSB-FGF-9 secondary mutant. The nucleotide sequences of the primers used in the above procedure are as follows:

The 77th phenylalanine codon, TTT, was substituted with TAT, the codon of tyrosine. The sense primer 5'-TTT CAT TTG GAA ATC TAT CCA AAT GGC ACG ATA -3 'and the antisense primer 3'-TAT CGT GCC ATT TGG ATA GAT TTC CAA ATG AAA-5 ';

AAG GAT CAT TCT CGA TAT GGC ATT CTG GAG TTT-3 'and antisense primer 3'-AAA CTC CAG AAT GCC ATA TCG AGA ATG ATC CTT -5 ';

GTC AGT GTA TTA TAT TTT GGT ATG AAT GAA AAA-3 'and antisense primer 3'-TTT TTC ATT CAT ACC AAA ATA TAA TCC when the CTT, the codon of 116th lysine, is substituted with TTT which is the codon of phenylalanine. GCT ATC-5 ';

When the CTT at codon 116 is substituted with ATG, which is a codon of mesaionine, the sense primer 5'-GAT AGC GGA TTA TAT ATG GGT ATG AAT GAA AAA-3 'and the antisense primer 3'-TTT TTC ATT CAT ACC CAT ATA TAA TCC GCT ATC -5 ';

CTG ACT CAG GAA TGT ACC TTT CGT GAA CAG TTT -3 'and antisense primer 3'-AAA CTG TTC ACG AAA GGT ACA TTC CTG TGT CAG -5 '; And

AAT TGT TAT AAC ACA TTT AGC TCC AAT CTG TAT -3 'and antisense primer 3'-ATA CAG ATT GGA GCT AAA TGT GTT ATA < RTI ID = 0.0 > CCA ATT -5 '.

GTC CGC CGC TAT TAT TTT GCG CTG AAC AAG GAT -3 'and the antisense primer 3'-ATC CTT GTT CAG CGC AAA ATA ATA GCG at the substitution of the 164th Bellin codon GTP with the codon TTT of phenylalanine GCG ACC -5 '.

ATG CTG GTT CAG CGC CAT ATA < SEP > ATT < SEP > ATT < SEP > ATT < SEP > ATT < SEP > ATA GCG GCG ACC -5 '.

AA4 AGG CAT CAA AAA TAT ACT CAC TTC CTG CCT -3 'and antisense primer 3'-AGG CAG GAA GTG AGT ATA TTT TTG ATG when the codon TTT of the 184th phenylalanine is substituted with TAT which is the codon of tyrosine. CCT TTT -5 '.

AAA TTT ACT CAC TTC CAT CCT CGT CCC GTT GAT -3 'and antisense primer 3'-ATC AAC GGG ACG AGG ATG GAA GTG AGT at the 188th lucyme codon CTG substitution with histidine codon CAT AAA TTT-5 '.

Each of the FGF-9 variants thus produced is as follows

Wherein the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A variant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A variant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A variant in which the 63rd and 96th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A variant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 63rd and 192th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 65th and 190th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine

Wherein the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 66th and 187th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine

Wherein the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted by tyrosine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid 116 of SEQ ID NO: 1 is substituted with phenylalanine.

A variant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 187th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine

Wherein the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted by tyrosine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the amino acid at position 166 of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 166th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine

Wherein the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 69th and 183rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

Wherein the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid in SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 72nd and 84th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid in SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 72nd and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 116 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 75th and 93rd amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 76th and 109th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

A variant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 80th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 82nd and 124th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 164th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 84th and 170th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 94th and 110th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 114th and 126th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine;

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A variant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 122nd and 175th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine;

1. A mutant comprising a mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid 92 of SEQ ID NO: 1 is substituted by tyrosine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 117th amino acid of SEQ ID NO: 1 is substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

Wherein the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 77 of SEQ ID NO: 1 is replaced with tyrosine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine and the 116th amino acid in SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the 116th amino acid of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine, and the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 164 of SEQ ID NO: 1 is substituted with mesaionine.

A mutant in which the 165th and 178th amino acids of SEQ ID NO: 1 are substituted with cysteine and the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

&Lt; Example 3: Production and purification of FGF-9 variants >

Each of the expression plasmids of FGF-9 variants was transformed into E. coli BL21 (DE3) in the same manner as in Example 2 to prepare an expression strain, and the expression strain was cultured in 500 ml of LB medium (LB / ampicillin) And purified in the same manner as in Example 1 to obtain FGF-9 of about 23 kDa in size. The amount of the mutant was varied depending on the mutant, and about 4 to 12 mg of FGF-9 was obtained depending on the mutant, and the purity was 98% or more.

Purification of the native form and variant can be done through ammonium sulphate and haparin affinity column. After final heparin affinity column purification, SDS-PAGE analysis was performed.

&Lt; Example 4: Structural analysis using CD of natural type and mutant FGF-9 >

The structure and thermal stability of the purified FGF-9 variants of Example 3 were determined by CD analysis using a J-810 spectrometer (JASCO). The native FGF-9 used was FGF-9 purified in Example 1. For structural analysis, each FGF-9 is dissolved in 20 mM sodium phosphate (pH 7.0), and the final concentration is adjusted to 0.1 mg / ml. The cell was immersed in a 0.1 cm cell and the band width was 1 nm, the response was 0.25 sec, the data pitch was 0.1 nm, the scanning speed was 20 nm / min, the cell length was 1 cm, the accumulation was 8 times, Respectively.

In order to analyze the thermal stability, Tm was compared with far-UV at 20 ° C and 95 ° C to determine the wavelength of 208 nm and the concentration was 0.1 mg / ml in 0.1 cm cell. The conditions were measured at 20 ° C to 95 ° C under the condition of 1 / min. And 57 ° C for the natural type Tm. The results are shown in Table 3.

No Cys mutantion location Change in Tm One Natural type 0 2 63 96 +0.5 3 63 192 +0.05 4 65 190 No change 5 66 187 +0.7 6 68 187 No change 7 68 166 +0.9 8 69 183 +0.2 9 72 84 +0.6 10 72 170 +0.8 11 75 93 +2 12 76 109 +2 13 80 126 +1.7 14 82 124 +1.3 15 84 170 No change 16 94 110 +1.3 17 114 126 No change 18 122 175 No change 19 134 117 +4.00 20 165 178 +1.00

Table 3 shows the results of measurement of the degree of structural change for the wild-type FGF-9 and FGF-9 variants and the fraction unfolded by temperature at a wavelength of 208 nm in the CD analysis of the thermal stability measurement experiment. When the folding-loosening phenomenon occurs, the structure changes in the vicinity of 208 nm. Using this, the Tm is measured within the range of 20 to 95 ° C, and the accurate Tm value is analyzed.

As a result, most of the structural changes showed the same structure as that of wild-type FGF-9 and there was no change, and the mutants added with disulfide bonds had no specific structure. Compared with wild-type FGF-9, which had a thermal stability of 57 ° C, Tm was increased to 4, 6, 15, 17, and 18, while Tm was increased in other mutants and thermal stability was significantly improved in mutant 19 there was. This means that the thermal stability is increased by artificially adding disulfide bonds by replacing the amino acid with cysteine. In addition, Tm changes were observed in the amino acid residues on the surface and the mutants added with the amino acid residue mutation for cavity stabilization (Tables 4 to 10).

Variant name Tm Variant name Tm Variant name Tm One 57 2 57.5 3 57.05 1_1 F184Y 57.3 2_1 F184Y 57.8 3_1 F184Y 57.45 1_2 F (77/92) Y 57.2 2_2 F (77/92) Y 57.7 3_2 F (77/92) Y 57.25 1_3 V135T 57.1 2_3 V135T 57.7 3_3 V135T 57.30 1_4 L188H 57.2 2_4 L188H 57.5 3_4 L188H 57.20 1_5 L116M 57.1 2_5 L116M 57.4 3_5 L116M 57.15 1_6 L116F 57.2 2_6 L116F 57.6 3_6 L116F 57.20 1_7 V164F 57.2 2_7 V164F 57.8 3_7 V164F 57.05 1_8 V164M 57.2 2_8 V164M 57.5 3_8 V164M 57.15 1_9 Y148F 57.2 2_9 Y148F 57.8 3_9 Y148F 57.20

Variant name Tm Variant name Tm Variant name Tm 4 57 5 57.7 6 57 4_1 F184Y 57.4 5_1 F184Y 58.2 6_1 F184Y 57.5 4_2 F (77/92) Y 57.6 5_2 F (77/92) Y 57.8 6_2 F (77/92) Y 57.4 4_3 V135T 57.2 5_3 V135T 57.7 6_3 V135T 57.2 4_4 L188H 57.1 5_4 L188H 58.0 6_4 L188H 57.3 4_5 L116M 57.0 5_5 L116M 57.7 6_5 L116M 57.5 4_6 L116F 57.5 5_6 L116F 57.7 6_6 L116F 57.2 4_7 V164F 57.4 5_7 V164F 57.9 6_7 V164F 57.3 4_8 V164M 57.5 5_8 V164M 57.8 6_8 V164M 57.2 4_9 Y148F 57.1 5_9 Y148F 57.8 6_9 Y148F 57.1

Variant name Tm Variant name Tm Variant name Tm 7 57.9 8 57.2 9 57.6 7_1 F184Y 58.4 8_1 F184Y 57.1 9_1 F184Y 57.9 7_2 F (77/92) Y 57.9 8_2 F (77/92) Y 57.2 9_2 F (77/92) Y 57.8 7_3 V135T 58.0 8_3 V135T 56.8 9_3 V135T 57.7 7_4 L188H 57.9 8_4 L188H 57.0 9_4 L188H 57.8 7_5 L116M 57.9 8_5 L116M 57.2 9_5 L116M 57.6 7_6 L116F 58.2 8_6 L116F 57.1 9_6 L116F 57.5 7_7 V164F 58.1 8_7 V164F 57.2 9_7 V164F 57.8 7_8 V164M 58.1 8_8 V164M 56.9 9_8 V164M 57.9 7_9 Y148F 57.6 8_9 Y148F 57.0 9_9 Y148F 57.9

Variant name Tm Variant name Tm Variant name Tm 10 57.8 11 59 12 59 10_1 F184Y 58.3 11_1 F184Y 59.6 12_1 F184Y 59.4 10_2 F (77/92) Y 58.0 11_2 F (77/92) Y 59.4 12_2 F (77/92) Y 59.5 10_3 V135T 57.9 11_3 V135T 59.3 12_3 V135T 59.4 10_4 L188H 58.1 11_4 L188H 59.4 12_4 L188H 59.4 10_5 L116M 57.8 11_5 L116M 59.0 12_5 L116M 59.0 10_6 L116F 57.9 11_6 L116F 58.9 12_6 L116F 59.1 10_7 V164F 58.0 11_7 V164F 59.4 12_7 V164F 59.2 10_8 V164M 57.9 11_8 V164M 59.2 12_8 V164M 59.1 10_9 Y148F 57.6 11_9 Y148F 59.1 12_9 Y148F 59.3

Variant name Tm Variant name Tm Variant name Tm 13 58.7 14 58.3 15 57 13_1 F184Y 59.2 14_1 F184Y 59 15_1 F184Y 57.0 13_2 F (77/92) Y 59.0 14_2 F (77/92) Y 58.4 15_2 F (77/92) Y 57.1 13_3 V135T 58.6 14_3 V135T 58.8 15_3 V135T 57.0 13_4 L188H 58.4 14_4 L188H 58.4 15_4 L188H 57.2 13_5 L116M 58.9 14_5 L116M 58.6 15_5 L116M 57.0 13_6 L116F 59.0 14_6 L116F 58.7 15_6 L116F 57.0 13_7 V164F 58.7 14_7 V164F 58.3 15_7 V164F 57.0 13_8 V164M 58.6 14_8 V164M 58.3 15_8 V164M 56.9 13_9 Y148F 58.9 14_9 Y148F 58.4 15_9 Y148F 57.3

Variant name Tm Variant name Tm Variant name Tm 16 58.3 17 57 18 57 16_1 F184Y 58.5 17_1 F184Y 57.0 18_1 F184Y 57.2 16_2 F (77/92) Y 58.9 17_2 F (77/92) Y 57.1 18_2 F (77/92) Y 57.3 16_3 V135T 58.4 17_3 V135T 57.4 18_3 V135T 57.1 16_4 L188H 58.6 17_4 L188H 57.3 18_4 L188H 57.2 16_5 L116M 58.5 17_5 L116M 57.2 18_5 L116M 57.4 16_6 L116F 58.3 17_6 L116F 57.3 18_6 L116F 57.1 16_7 V164F 58.9 17_7 V164F 57.1 18_7 V164F 57.3 16_8 V164M 58.4 17_8 V164M 57.3 18_8 V164M 57.2 16_9 Y148F 58.3 17_9 Y148F 57.1 18_9 Y148F 57.0

Variant name Tm Variant name Tm 19 61 20 58 19_1 F184Y 61.5 20_1 F184Y 58.4 19_2 F (77/92) Y 61.5 20_2 F (77/92) Y 58.2 19_3 V135T 61.4 20_3 V135T 58.1 19_4 L188H 61.4 20_4 L188H 58.4 19_5 L116M 61.2 20_5 L116M 58.4 19_6 L116F 61.4 20_6 L116F 58.6 19_7 V164F 61.3 20_7 V164F 58.4 19_8 V164M 61.0 20_8 V164M 58.0 19_9 Y148F 61.2 20_9 Y148F 58.2

The mutant FGF-9 was selected and the cell proliferation assays were performed using the results of the structural and Tm analyzes using the solubility and circular dichroism among the wild-type FGF-9 and mutants. Cell proliferation assays were performed with BALB-3T3 cell lines. BALB-3T3 cells were maintained in DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. BALB-3T3 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well culture plate. BALB-3T3 cells cultured for 24 hours were incubated with serum-free DMEM medium, treated with the sample solution in DMEM medium containing 0.5% FBS, and cultured for 72 hours. After incubation, 10 μl of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide] solution was added and reacted for 2 hours. &Lt; / RTI > Absorbance was measured at 540 nm using a spectrophotometer. Sensitivity to the drug was compared by the percentage of absorbance of the untreated well (control) in the drug treated wells. As shown in FIG. 2, the FGF-9 mutants have similar cell proliferative capacity to that of native FGF-9.

Example 6 Quantitative Analysis of Proteins by Incubation of Natural and Mutant FGF-9

In order to confirm the stability of FGF-9 mutants substituted with cysteine in aqueous solution during long-term storage, 25 ℃ incubation test was performed. In the PBS (phosphate buffer saline) state most similar to the human body, the native FGF-9 and mutants were dissolved at 0.5 mg / ml and incubated in a water bath at 25 ° C. The cells were sampled at 7 days and centrifuged at 13000 rpm for 15 minutes at 4 ° C to obtain the supernatant. The results corresponding to Fig. 3 were obtained.

An incubation test at 50 ° C was carried out in the same manner as above. In the case of PBS (phosphate buffer saline), which is most similar to the human body, the native FGF-9 and mutants were dissolved at 0.5 mg / ml and incubated in a water bath at 50 ° C. The cells were sampled at 1 day and centrifuged at 13,000 rpm for 15 minutes at 4 ° C to obtain cell supernatants. The corresponding results of Fig. 4 were obtained.

&Lt; 110 > PnP Biopharm Co., Ltd. <120> A human fibroblast growth factor-9 mutant with high stability and use of the same <130> P16-0002 <160> 30 <170> Kopatentin 2.0 <210> 1 <211> 208 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 1 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95 Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205 <210> 2 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 2 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgctgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctgtgctt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 3 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 3 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgctgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt ccctgcgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 4 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 4 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggtgccttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgccttgc cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 5 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 5 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaatgcta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactg cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 6 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 6 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactg cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 7 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 7 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgtgcaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 8 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 8 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgctgcaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaatgct ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 9 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 9 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttgccatt tggaaatctt tccaaatggc 240 acgatacagt gcactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 10 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 10 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttgccatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggattgc acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 11 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 11 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tgtgcatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattctgca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 12 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 12 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaatgctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgttgcgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 13 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 13 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaattgc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatactgctc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 14 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 14 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgtgccagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat gctacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 15 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 15 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagt gcactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggattgc acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 16 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 16 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggct gcctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggttgc gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 17 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 17 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat gctatcttgg tatgaatgaa 360 aaaggcgaat tatactgctc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 18 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 18 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaatgcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aatgcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 19 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 19 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatctttg catgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 20 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 20 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tctgcctgaa caaggatggt acgccgagag aaggcaccag atgcaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 21 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 21 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatcta tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 22 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 22 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgatatggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 23 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 23 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatattttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 24 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 24 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatatggg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 25 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 25 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtacctttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 26 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 26 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atttagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatg tcgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 27 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 27 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattatt ttgcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 28 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 28 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattata tggcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 29 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 29 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattata tggcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat atactcactt cctgcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624 <210> 30 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 30 atggcgccgc taggggaggt agggaattac ttcggagtcc aggacgcagt gccttttggc 60 aacgtgcctg ttttaccggt tgacagccca gtgcttctct ccgaccacct gggtcaaagt 120 gaagcaggcg gtctgccgcg tgggcccgcc gtaaccgatc tggatcattt aaaaggaata 180 ctgcgccgcc ggcaacttta ttgccggaca ggttttcatt tggaaatctt tccaaatggc 240 acgatacagg ggactcgtaa ggatcattct cgattcggca ttctggagtt tatctctatt 300 gctgtgggtt tggttagtat tcgtggtgta gatagcggat tatatcttgg tatgaatgaa 360 aaaggcgaat tatacggatc agaaaaactg acacaggaat gtgtttttcg tgaacagttt 420 gaagaaaatt ggtataacac atacagctcc aatctgtata aacatgtgga caccggtcgc 480 cgctattata tggcgctgaa caaggatggt acgccgagag aaggcaccag aaccaaaagg 540 catcaaaaat ttactcactt ccatcctcgt cccgttgatc cggataaggt tccagagttg 600 tataaagata ttctctcaca gtcg 624

Claims

delete

A mutant in which the 63rd and 96th residues of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 63rd and 192th motifs of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 65th and 190th nucleotides of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 66th and 187th of SEQ ID NO: 1 are substituted with cysteine,
A mutant obtained by substituting cysteine at position 187 of SEQ ID NO: 1,
A mutant in which the 166th position of SEQ ID NO: 1 is substituted with cysteine,
A mutant in which the 69th and 183rd orders of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 72nd and 84th residues of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 72nd and 170th motifs of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 75th and 93rd residues of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 76th and 109th nucleotides of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 80th and 126th residues of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 82nd and 124th residues of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 84th and 170th nucleotides of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 94th and 110th motifs of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which the 114th and 126th motifs of SEQ ID NO: 1 are substituted with cysteine,
A mutant in which positions 122 and 175 of SEQ ID NO: 1 are substituted with cysteine,
A mutant substituted with the 117th cysteine of SEQ ID NO: 1, and
A mutant of the high-stability fibroblast growth factor (FGF-9) selected from the group consisting of mutants substituted with 165th and 178th cysteines of SEQ ID NO: 1.

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 63 and 96 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 63 and 192 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 65 and 190 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 66 and 187 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is selected from the group consisting of SEQ ID NO:
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at position 166 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

The method of claim 2 wherein the variant is substituted in the 69th and 183rd the cysteine of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 72 and 84 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

The method of claim 2 wherein the variant is substituted in the 72nd and 170th cysteine of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 75 and 93 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is substituted with threonine, the amino acid at position 148 of SEQ ID NO: 1 is substituted with phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which the amino acid at position 184 is substituted with tyrosine, or the amino acid at position 188 of SEQ ID NO: 1 is substituted with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 76 and 109 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 80 and 126 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 82 and 124 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is replaced with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 84 and 170 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

3. The method of claim 2, wherein the mutant is substituted with cysteine at positions 94 and 110 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 114 and 126 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

3. The method according to claim 2, wherein said mutant is substituted with cysteine at positions 122 and 175 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

[3] The method according to claim 2, wherein the mutant is substituted with cysteine at position 117 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

3. The method according to claim 2, wherein the mutant is substituted with cysteine at positions 165 and 178 of SEQ ID NO: 1,
The amino acid at position 77 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 92 of SEQ ID NO: 1 is substituted with tyrosine, the amino acid at position 116 of SEQ ID NO: 1 is substituted with phenylalanine, 1, the amino acid at position 135 of SEQ ID NO: 1 is replaced with threonine, the amino acid at position 148 of SEQ ID NO: 1 is replaced by phenylalanine, the amino acid at position 164 of SEQ ID NO: 1 is substituted with phenylalanine, A mutant in which amino acid 184 of SEQ ID NO: 1 is substituted with tyrosine, or amino acid 188 of SEQ ID NO: 1 is replaced with histidine.

delete

The method according to any one of claims 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 27 29, 31, 33, 35, 37, 39, and 41. The gene encoding the mutant of any of claims 29, 31, 33, 35, 37, 39,

43. The gene according to claim 43, wherein said gene consists of a DNA sequence selected from the group consisting of SEQ ID NOS: 2 to 30.

43. An expression vector comprising the gene of claim 43.

45. A transformant transformed by the expression vector of claim 45.

delete