KR101891678B1

KR101891678B1 - A Highly-Stable Mutant of Basic Fibroblast Growth Factor with Modified N-Terminal Amino acid Region, And Uses Thereof

Info

Publication number: KR101891678B1
Application number: KR1020160092598A
Authority: KR
Inventors: 신항철; 오종광; 박연희
Original assignee: (주)피앤피바이오팜
Priority date: 2016-07-21
Filing date: 2016-07-21
Publication date: 2018-08-27
Also published as: KR20180010468A; WO2018016814A1

Abstract

The present invention relates to a method for increasing productivity by inhibiting degradation in a production process using a microorganism through modification of an N-terminal amino acid sequence of a high-stability bFGF. More specifically, mutants derived from deletion and insertion of N-terminal mutants in a highly stable bFGF mutant in which two or more amino acids in the amino acid sequence of SEQ ID NO: 1 are substituted with serine and at least one amino acid is substituted with cysteine, A DNA base sequence encoding the bFGF mutant, an expression vector containing the DNA base sequence, a transformant transformed with the expression vector, a method for producing the bFGF mutant, and a composition comprising the bFGF mutant as an active ingredient do. According to the present invention, the bFGF mutant of the present invention is excellent in heat stability and stability in an aqueous solution state, and thus it is possible to produce functional cosmetic products and skin inflammation treatment products which do not lose activity unlike conventional natural bFGF products during distribution and storage .

Description

[0001] The present invention relates to a high-stability fibroblast growth factor variant having a modified N-terminal amino acid region and a use thereof.

The present invention relates to a high-stability fibroblast growth factor variant having a modified N-terminal amino acid site and use thereof, and more particularly to a high-stability fibroblast growth factor (bFGF) The present invention relates to the production of pure single-component, high-stability bFGF by inhibiting the disappearance of amino acid at the N-terminal which may occur in the production process using microorganisms through modification of the sequence.

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, bFGF is composed of 154 amino acids and has a molecular weight of 17,123 dalton. It plays an important role in development, angiogenesis and wound healing. bFGF is known as a potent mediator of wound healing, angiogenesis, and nervous system growth as mitogen and chemotactic factor.

However, the growth factors present in these blood and tissues are known to have a very short half-life of about several minutes. In particular, bFGF has four cysteine residues that do not form a disulfide bond There is a problem that it is greatly influenced by its stability.

In addition, the bioavailability of protein therapeutics such as bFGF is often limited by short half-lives in blood and susceptibility to proteolytic enzymes, hindering maximum clinical efficacy. In order to improve bFGF more effectively, physico-chemical stability in vitro as well as stability in the body should be improved, leading to increased use in the manufacture, storage and distribution of medicines and cosmetics. In order to overcome such a problem, a high-stability bFGF (designated HsbFGF) in 2016 has been developed in P. pneumoniae (KR 10-2015-0078930 / PCT-KR2015-007734).

However, when high-stability bFGF is produced using E. coli, the N-terminal of bFGF is partially hydrolyzed by an aminopeptidase, resulting in the loss of some amino acids at the N-terminal portion of bFGF. As a result, the highly purified bFGF final purified product has a problem in which individuals having different N-terminal ends are mixed with each other and further separation is difficult.

That is, the N-terminal heterogeneity problem is mistaken for impurities, that is, it is perceived as not a single component, which hinders the production and quality control of recombinant proteins and is recognized as a low-purity product in the license process It is a task that must be done.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a high-stability bFGF N-terminal mutant capable of successfully inhibiting the disappearance of N-terminal amino acid.

Another object of the present invention is to provide a DNA base sequence encoding a bFGF mutant.

Still another object of the present invention is to provide an expression vector comprising the DNA base sequence.

It is still another object of the present invention to provide a transformant transformed with the expression vector.

It is still another object of the present invention to provide a method for producing a high-stability bFGF mutant.

It is still another object of the present invention to provide a cosmetic composition for improving skin condition comprising a high-stability bFGF mutant as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating skin diseases, which comprises a high-stability bFGF mutant as an active ingredient.

In order to attain the above object, the present invention provides a high-stability bFGF having two or more amino acids in the amino acid sequence of SEQ ID NO: 1 substituted with serine, one or more amino acids substituted with cysteine, and one amino acid on the surface thereof substituted with tyrosine Provide variants.

More preferably, a deletion mutation is introduced into two amino acid residues at the N-terminal region of the existing high-stability bFGF mutant, and methionine is inserted to prevent N-terminal hydrolysis which may occur immediately after the recombinant protein expression, .

As used herein, the term bFGF is a basic protein with a molecular weight of about 18 kDa (pI 9.58) which is mainly secreted in the pituitary gland and is known to promote the growth of a variety of mesenchymal stem cells. Also, it is a protein that promotes the growth of endothelial cells and smooth muscle cells, and exhibits excellent effects in trauma treatment and vasculogenesis. It increases the synthesis of collagen and elastin, thereby maintaining skin elasticity, , And is known to perform the healing action.

The mutant of the present invention can be prepared by selecting a site which is not related to the active site of bFGF through the homology alignment method of the tertiary structure of bFGF and species and the computer protein molecular modeling, As a mutant, the cysteine amino acid residue forming a disulfide bond with bFGF and another bFGF is substituted with a serine residue having a similar structure, thereby increasing stability against precipitation due to surface disulfide bonds. It is also characterized in that stability is improved by reducing loop entropy by additionally producing a disulfide bond by substituting one residue near the loop in bFGF with cysteine. In addition, stability is improved by substituting tyrosine for a histidine residue in bFGF to increase hydrogen bond and van der Waals interaction to stabilize the cavity structure inside the protein structure.

According to a preferred embodiment of the present invention, the amino acid substituted with serine is the 68th cysteine and the 86th cysteine in the amino acid sequence of SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the amino acid substituted with cysteine is the 25th lysine in the amino acid sequence of SEQ ID NO: 1; The 33rd isoleucine in the amino acid sequence of SEQ ID NO: 1; 39th valine in the amino acid sequence of SEQ ID NO: 1; The 49th histidine in the amino acid sequence of SEQ ID NO: 1; The 51st lysine in the amino acid sequence of SEQ ID NO: 1; The 74th alanine in the amino acid sequence of SEQ ID NO: 1; The 75th methionine in the amino acid sequence of SEQ ID NO: 1; The 116th alanine in the amino acid sequence of SEQ ID NO: 1; The 66th glycine in the amino acid sequence of SEQ ID NO: 1; The 67th valine in the amino acid sequence of SEQ ID NO: 1; The 69th alanine in the amino acid sequence of SEQ ID NO: 1; The 81st leucine in the amino acid sequence of SEQ ID NO: 1; The 83rd alanine in the amino acid sequence of SEQ ID NO: 1; The 107th serine in the amino acid sequence of SEQ ID NO: 1; The 135th alanine in the amino acid sequence of SEQ ID NO: 1; The 136th isoleucine in the amino acid sequence of SEQ ID NO: 1; The 137th leucine in the amino acid sequence of SEQ ID NO: 1; And the 143th alanine in the amino acid sequence of SEQ ID NO: 1, more preferably at least the 39th amino acid in the amino acid sequence of SEQ ID NO: 1; The 49th histidine in the amino acid sequence of SEQ ID NO: 1; The 51st lysine in the amino acid sequence of SEQ ID NO: 1; The 74th alanine in the amino acid sequence of SEQ ID NO: 1; The 75th methionine in the amino acid sequence of SEQ ID NO: 1; And the 116th alanine in the amino acid sequence of SEQ ID NO: 1, and most preferably the 74th alanine in the amino acid sequence of SEQ ID NO: 1.

As a further variant, a variant obtained by substituting tyrosine residue of histidine residue 49, which is a residue exposed on the surface of a protein, with a mutant in which the 74th alanine is substituted with cysteine is the most preferable variant.

That is, the bFGF mutant of the present invention is a mutant of the 68th and 86th amino acids of the mutant (SEQ ID NO: 1) in which methionine is added as a starting sequence after removing two N-terminal amino acid sequences from the wild-type The cysteine is all substituted with serine, the 49th histidine is substituted with tyrosine, alanine, which is the 74th amino acid residue, is further substituted by cysteine to form a disulfide bond in the molecule, and the remaining amino acid sequence is the same as the native amino acid sequence, Lt; / RTI >

The bFGF mutant of the present invention is a mutant produced by inducing a deletion mutation of the N-terminal sequence in the sequence of the above-mentioned existing HsbFGF (KR 10-2015-0078930 / PCT-KR2015-007734).

More preferably, proline and alanine, the first and second sequences of the N-terminal of the existing HsbFGF, are introduced as a start sequence by inducing a deletion mutation and inserting methionine.

The bFGF mutant of the present invention increases the stability to heat as compared with the wild type while maintaining the protein activity. As shown in the following Experimental Example 2, the bFGF mutant has the activity equivalent to that of the wild type, and the stability against heat is also remarkably increased.

mutants K (sbFGF, K74) of the present invention in which 68 and 86 amino acids are substituted with serine, 74 amino acid is replaced with cysteine, and disulfide bonds are induced, and mutant K is substituted with tyrosine 49 of histidine Mutant O (HsbFGF) had improved thermal stability over the wild type control.

According to another aspect of the present invention, the present invention provides a DNA base sequence (SEQ ID NO: 2) encoding the bFGF mutant and an expression vector comprising the same.

The expression vector of the present invention can be prepared by inserting the gene of bFGF 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 in which a bFGF gene is inserted into a pET21a vector was prepared and named "pSSB-bFGF" (FIG.

According to another aspect of the present invention, the present invention provides a transformant which is a host cell transformed with said expression vector.

The bFGF mutant of the present invention can be produced by a method of expressing a bFGF mutant by transforming a host cell with a vector containing a gene encoding a bFGF mutant produced by a site specific mutagenesis method or the like, .

As a DNA sequence coding for the bFGF mutant, the N-terminal proline and alanine are removed from native bFGF, the methionine is inserted thereinto, the 68th and 86th codons are substituted with serine coding codons, The codon is replaced with a codon that codes for cysteine. In addition, mutant O (HsbFGF) substituted with tyrosine at position 49 of mutant K improved the thermal stability of the wild type and mutant K (sbFGF).

On the other hand, it is well known that a nucleotide sequence of a DNA encoding the same amino acid sequence may be different because a plurality of codons encoding one amino acid are present due to degeneracy of codons.

The DNA coding for such bFGF mutant can be chemically synthesized or prepared by producing a native bFGF cDNA and using site-directed mutagenesis based thereon.

The prepared DNA encoding the bFGF mutant of the present invention can be expressed using any suitable prokaryotic or eukaryotic expression system (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd < RTI ID = 0.0 > Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor 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 for non-glycosylated bFGF variants, and suitable vectors that can be used for expression in E. coli include, (Durand et al., Eds.), Pp. 680-697 ("Proced. 8th Int. Biotechnology Symposium ", Soc. Frac. De Microbiol., Paris, , 1988).

Transformation of host cells by the vectors described above can be carried out by any of the conventional methods (Sambrook et al., Molecular Cloning, A Laboratory Manual, 1989; Ito et al., J. Bacteriol. 153: 263 , 1983).

When transforming E. coli, 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 bFGF mutant comprising the steps of:

(a) culturing the transformant; And

(b) separating the mutant from the culture obtained in the step (a).

According to a preferred embodiment of the present invention, step (b)

(c) disrupting the transformant and removing aggregates;

(d) separating and purifying the aggregate-removed supernatant using ion exchange resin chromatography; And

(e) separating and purifying the high-stability bFGF mutant 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 bFGF mutant after culturing the transformed host cell can be carried out by various methods known in the art or by using them in combination. For example, the bFGF mutant expressed in the transformed E. coli cells can be recovered from the cell culture or after the disruption of the cells by suitable methods known in the field of protein chemistry.

Preferably, in order to purify the bFGF mutant, the culture medium of the recombinant E. coli cells is centrifuged to harvest the cells, and the harvested cells are suspended in the lysozyme-added buffer solution and ultrasonicated. The cell lysate is centrifuged to remove insoluble granular aggregates. The supernatant, from which the aggregates have been 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 bFGF mutant.

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

As demonstrated in the following examples, the high-stability bFGF variants of the present invention have the same activity as native bFGF, 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 compositions of the present invention are useful for the treatment and / or prophylaxis of skin inflammation, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrhoeic dermatitis, chronic simplex poisoning, biliary cirrhosis, deprivation dermatitis, It is used to prevent or treat skin 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 closure windows include contusion or bruise and examples of open windows include abrasion, laceration, avulsion, penetrated wound, and gun-shot wound .

The composition of the present invention comprises (a) a pharmaceutically effective amount of the above-mentioned bFGF mutant 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 bFGF variants described above.

The pharmacologically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one which is usually used in the production, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, It is not. 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 0.001-100 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 bFGF mutant as an active ingredient.

As demonstrated in the following examples, the high-stability bFGF variants of the present invention have the same activity as native bFGF, 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 a bFGF mutant of the present invention as described above; 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 a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is 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 is selected from 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 ingredients contained in the cosmetic composition of the present invention include, in addition to the bFGF mutant and the carrier component as the active ingredient, components commonly used in cosmetic compositions and include conventional components such as antioxidants, stabilizers, solubilizers, vitamins, Phosphorous assistant.

Since the compositions of the present invention include the above-described highly stable bFGF 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 novel HsbFGF mutant produced by the modification of the N-terminal has excellent thermal stability and stability in the aqueous solution state, so that it does not lose its activity differently from the conventional natural bFGF product during distribution and storage, It is made of a pure single substance and can be used as a material for functional cosmetics and as a treatment for skin wound.

Figure 1 shows an overview of the assembly of plasmid pSSB-bFGF.
FIG. 2 is an analysis using SDS-PAGE after final purification of a native bFGF, a conventional K75 (sbFGF), and a novel HsbFGF (mutant O) developed in the present invention.
FIG. 3 shows the difference in melting temperature (Tm), which is an index of the thermal stability of the native bFGF and K74 (mutant K), the existing HsbFGF (mutant of the present invention) and the novel HsbFGF (mutant O).
Fig. 4 shows the stability comparison results of the native bFGF, the existing HsbFGF and the new HsbFGF (mutant O) after 5 days incubation at 50 ° C in the phosphate buffer solution.
FIG. 5 shows the stability comparison results of the native bFGF, the existing HsbFGF and the novel HsbFGF (mutant O) after incubation at 60 ° C for 5 days in the phosphate buffer solution.
FIG. 6 shows the results of comparing the activity of wild-type bFGF with the novel HsbFGF (mutant O) of the present invention and the existing HsbFGF activity.
7 shows (a) the N-terminal analysis result of the novel HsbFGF (mutant O) of the present invention and (b) the N-terminal analysis result of the existing HsbFGF.
FIG. 8 shows the results of incubation of native bFGF with a conventional HsbFGF mutant and novel HsbFGF (mutant O) at 37 ° C for 1 month through concentration determination.
FIG. 9 shows the results of (a) HPLC and (b) SDS-PAGE analysis of the incubation results of natural type bFGF, K74 (mutant K) and novel HsbFGF (mutant O) of the present invention at 37 ° C for 100 days.
10 shows the result of toxicity test using L929 cells of new HsbFGF (mutant O).

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 Mold making (construction)

BL21 (DE3) was purchased from Novagen for protein expression vector pET21a (Fig. 1A) and E. coli for expression, and Top10 was used for 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. The Ex taq DNA polymerase used in the PCR was a product of Takara Co. and the pfuUltra HF DNA polymerase 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 produced by Cosmojin Tech Co., Ltd., and DNA sequencing was also performed by Cosmojin Tech Co., Ltd.

Protein expression

The expression derivative, IPTG (isopropyl-1-thio-β-D-galactopyranoside) and the antibiotic ampicillin and chloramphenicol were all 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-sepharose and heparin-affinity columns.

FPLC

FPLC used GE UPC-800.

CD (Circular dichroism )

The CD was a J-810 spectropolarimeter from Jasco.

Homology modelling (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 for the plotting program to measure the disposable distance.

Point mutation

In order to increase the stability of bFGF, the amino acid part to be changed through the structure of the protein (PDB: 4FGF) and the molecular model method was found. Using the following primer (Example 2), pfuUltra HF DNA polymerase was used for quickchange mutagenesis Lt; / RTI > In order to remove the natural bFGF template used, the DpnI reaction was carried out to transform Top10 and the mutant was identified by sequencing.

molecule modelling (Molecular modeling)

A candidate group capable of disulfide bonding was set using 1BLA (NMR), which is a structure of proteins registered in the PDB. Using a protein contact map visualization program, the residues with C-alpha carbon distance of less than 7 Å and C-beta carbon distance of 5 Å were analyzed by 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 rearranged with the native bFGF to obtain a structure having a value of RMSD of 0.5 or less.

CD (Circular dichroism )

For structural analysis and Tm measurement of native bFGF and mutants, make bFGF constant to 0.2 mg / ml. Then, in a 0.1 cm cell, 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 pathlength length was 1 cm, the accumulation was 8 times, Respectively. The melting temperature was 0.2 mg / ml in a 0.1 cm cuvette at 205 nm wavelength at 20 ℃ and 95 ℃. The conditions were measured at a rate of 1 占 폚 / min from 20 占 폚 to 95 占 폚.

Cell proliferation assay and toxicity test (cell proliferation assay)

Experiments were carried out using the cell proliferation ability to confirm that the produced natural type bFGF and the mutant actually show activity. The NIH-3T3 and L929 cells used in the experiments were maintained in DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. 2 × 10 ³ cells / well of NIH-3T3 cells were seeded in a 96-well culture plate. NIH-3T3 cells cultured for 24 hours were treated with serum-free DMEM medium and then treated with the sample solution in DMEM medium containing 0.5% FBS for 72 hours after starvation. 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. 100 μl of DMSO was added to formazan crystal &Lt; / RTI > Absorbance was measured at 540 nm using a spectrophotometer. The susceptibility to the drug was compared with the percentage of absorbance of the wells (control) not treated with the drug in the treated wells.

Incubation test

In order to confirm the degree of preservation at room temperature, incubation test of wild type bFGF and mutant was carried out. Each native bFGF and its mutants were dissolved at 0.5 mg / ml in 1X PBS (pH 7.2) and incubated at 37 ° C, 50 ° C and 60 ° C. Sampling was carried out for 24 hours and centrifugation was carried out at 4 ° C for 15 minutes at 13000 rpm. Only the supernatant was collected and quantitated by nano-drop and HPLC analysis was carried out.

&Lt; Example 1: Construction and purification of pSSB-bFGF plasmid containing human bFGF cDNA >

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

Sense primer: 5'-GGCGGGCATATGTTGCCCGAGG-3 '

Antisense primer: 3'-CCTCGGGCAACATATGCCCGCC-5 '.

The bFGF portion of FIG. 1B was amplified using the primers described above, and 1 μg of the amplified DNA fragment was dissolved in 50 μl of TE (pH 8.0) solution. Then, 2 units of NdeI (NEB) and 2 units of BamHI (NEB), and reacted at 37 ° C for 2 hours to obtain 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-bFGF. E. coli BL21 (DE3) was transformed by heat shock with the expression plasmid thus prepared. Colonies resistant to ampicillin generated in the solid medium after transformation were selected and inoculated into 10 ml of LB medium (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 12 hours or more. Then, the cells were transferred to 500 ml of LB medium (LB / ampicillin), and IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added at a final concentration of 0.5 mM at OD ₆₀₀ 0.4-0.5. 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. Then, SP and heparin columns were purified using FPLC. The fractions containing bFGF were identified by SDS-PAGE for each fraction and then quantified by Bradford assay. As a result, 10 to 18 mg of bFGF was obtained.

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

The pSSB-bFGF plasmid of natural type was used as a template and pfuUltra By using HF DNA polymerase, pSSB-bFGF mutant plasmids were prepared by PCR using two complementary primers corresponding to each mutant. The mutant plasmid was identified. The base sequence of the primers used was as follows:

TCT ATC AAA GGA GTG TCT GCT AAC CGT TAC CTG-3 'and antisense primer 3'-CAG GTA ACG GTT AGC AGC CAC TCC TTT GAT AGA-5 ';

TTT CTG GCT TCT AAT TCT GTT ACT GAT GAG TGT-3 'and antisense primer 3'-ACA CTC ATC CGT AAC AGA TTT AGA AGC CAG TAA-5 ';

GCT AAC CGT TAC CTG TGC ATG AAG GAA GAT GGA-3 'and antisense primer 3'-TCC ATC TTC CTT CAT GCA CAG GTA ACG at the substitution of TCT, the codon of 74th alanine, GTT AGC-5 ';

AAG CGG CTG TAC TGC TGC AAC GGG GGC TTC TTC-3 'and antisense primer 3'-GAA GAA GCC CCC GTT GCA GCA GTA CAG at the time of substitution with the codon of cysteine TGC at the 25th lysine codon CCG CTT-5 ';

When ATC, which is the codon of the 33rd isoleucine, is substituted with TGC, which is the codon of cysteine, the sense primer 5'-GGC TTC TTC CTG CGC TGC CAC CCC GAC GGC CGA-3 'and antisense primer 3'-TCG GCC GTC GGG GTG GCA GCG CAG GAA GAA GCC-5 ';

CTC CCG GAC CCC GTC GCA TCG GCC GTC GTC GGA CGG GAG-3 'and sense primer 3'-CTC CCG GAC GTC GTC GTC CGC GCC GTC GGA CGG GAG-3' at the substitution of the 39th valine codon GTG with the codon TGC of the cysteine sense primer 5'- CAC CCC GAC GGC CGA TGC GAC GGG GTC CGG GAG- GGG GTG-5 ';

GAG AAG AGC GAC CCT TGC ATC AAG CTA CAA CTT-3 'and antisense primer 3'-AAG TTG TAG CTT GAT GCA AGG GTC GCT CTT CTC-3' -5 ';

5'-AGC GAC CCT CAC ATC TGC CTA CAA CTT CAA GCA-3 'and antisense primer 3'-TGC TTG AAG TTG TAG GCA GAT GTG AGG at the substitution of the codon AAG of the 51st lysine with TGC, GTC GCT-5 ';

ATP CGT TAC CTG GCT TGC AAG GAA GAT GGA AGA-3 'and antisense primer 3'-TCT TCC ATC TTC CTT GCA AGC CAG GTA when ATG, the codon of the 75th methionine, was substituted with TGC, the codon of cysteine ACG GTT-5 ';

ACC AGT TGG TAT GTG TGC CTG AAG CGA ACT GGG-3 'and antisense primer 3'-CCC AGT TCG CTT CAG GCA CAC ATA CCA at the substitution of the 116th alanine codon GCA for the cysteine codon TGC ACT GGT-5 ';

GTG TCT ATT AAA TGC GTG TCT GCT AAC CGT-3 'and antisense primer 3'-ACG GTT AGC AGC CAC GCA TTT GAT AGA at the substitution of GGA, the codon of the 66th glycine, with TGC, CAC AAC-5 ';

GTG TCT ATT AAA GGA TGC TCT GCT AAC CGT TAC-3 'and antisense primer 3'-GTA ACG GTT AGC AGA GCA TCC TTT GAT AGA CAC-5 ';

ATC AAA GGA GTG TCT TGC AAC CGT TAC CTG GCT-3 'and antisense primer 3'-AGC CAG GTA ACG GTT GCA AGA CAC TCC when the GCT, the 69th alanine codon, was substituted with TGC, the codon of cysteine TTT GAT-5 ';

AAG GAA GAT GGA AGA TGC CTG GCT TCT AAA TCT-3 'and antisense primer 3'-AGA TTT AGA AGC CAG GCA TCT TCC ATC at the time of substitution with the codon TGC of cysteine TTC CTT-5 ';

GAT GGA AGA TTA CTG TGC TCT AAA TCT GTT ACG-3 'and antisense primer 3'-CGT AAC AGA TTT AGA GCA CAG TAA TCT when the codon of 85th alanine is substituted with TGC, TCC ATC-5 ';

TAC AAT ACT TAC CGG TGC AGG AAA TAC ACC AGT-3 'and antisense primer 3'-ACT GGT GTA TTT CCT GCA CCG GTA AGT-3' at the substitution of TCA, the 107th serine codon, with TGC, ATT GTA-5 ';

GGCCGTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG AGG TCC-5 ';

CCT ATG-3 'and antisense primer 3'-CAT TGG AAG AAA AAG GCA AGC TTT < RTI ID = 0.0 > CTG CCC AGG-5 ';

GGG CAG AAA GCT ATA TGC TTT CTT CCA ATG TCT-3 'and antisense primer 3'-AGA CAT TGG AAG AAA GCA TAT AGC TTT when the CTT of 137th lysine was substituted with TGC, the codon of cysteine CTG CCC-5 '; And

TCT CCA ATG TCT TGC AAG AGC TGA TGA-3 'and antisense primer 3'-TCA TCA GCT CTT GCA AGA CAT TGG AAG AAA -5 '.

GAG AAG AGC GAC CCT TAT ATC AAG CTA CAA CTT -3 'and antisense primer 3'-AAG TTG TAG CTT GAT ATA AGG GTC GCT CTT CTC-5 '.

&Lt; Example 3: Production and purification of bFGF mutants >

Expression of bFGF Mutants The cells were cultured in 500 ml of LB medium (LB / ampicillin) and purified in the same manner as in Example 1 to obtain bFGF of about 18 KDa in size. The amount of mutant was varied depending on the mutant, and about 4 ~ 12 mg of bFGF was obtained depending on the mutant, and the purity was 98% or more.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 33rd isoleucine and 66th glycine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 33rd isoleucine and 69th alanine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 33rd isoleucine and 83rd alanine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 39th valine and 81st leucine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 39th valine and 83rd alanine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 49th histidine and 68th cysteine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 51st lysine and 67th valine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 75th methionine and the 107th serine are substituted with cysteine.

A mutant in which the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 116th alanine and 135th alanine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 116th alanine and 136th isoleucine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 74th alanine is substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 25th lysine and 86th cysteine are substituted with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 137th leucine is replaced with cysteine.

Wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, and the 51st lysine and 143th alanine are substituted with cysteine.

A mutant in which the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, the 74th alanine is substituted with cysteine, and the 49th histidine is substituted with tyrosine.

The purified form of the native form and variant can be purified through SP-sepharose and heparin affinity columns. After final heparin-affinity column purification, SDS-PAGE analysis was performed.

As shown in FIG. 2, dimer and trimer were observed in the native form, whereas it was confirmed that the mutant (HsbFGF) existed in the form of a single band in the monomer size. This is the data showing that the dimer and trimer without activity are completely removed and exist in the monomer state.

EXPERIMENTAL EXAMPLE 1: Structural analysis of natural and mutant bFGF using circular dichroism.

The structure and thermal stability of the purified bFGF variants of Example 3 were measured by circular dichroism analysis using J-810 spectropolarimeter (JASCO). The native bFGF used in Example 1 was purified bFGF. For structural analysis, each bFGF 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.

To analyze 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 a 0.1 cm cuvette. The conditions were measured at a rate of 1 占 폚 / min from 20 占 폚 to 95 占 폚. The results are shown in Table 1.

Variant bFGF designation Structural change (Tm) Variant bFGF designation Structural change (Tm) Variant bFGF designation Structural change (Tm) The bFGF
(SEQ ID NO: 1) -
(57.5 DEG C) Mutant A
C68S, C86S
I33C, G66C
(SEQ ID NO: 3) decrease
48 ° C Mutant B
C68S, C86S
I33C, A69C
(SEQ ID NO: 4) No change Variant C
C68S, C86S
I33C, A83C
(SEQ ID NO: 5) No change Variant D
C68S, C86S
V39C, L81C
(SEQ ID NO: 6) No change Mutant E
C68S, C86S
V39C, A83C
(SEQ ID NO: 7) No change Mutant F
C68S, C86S
H49C
(SEQ ID NO: 8) No change Mutant G
C68S, C86S
K51C, V67C
(SEQ ID NO: 9) No change Mutant H
C68S, C86S
M75C, S107C
(SEQ ID NO: 10) No change Mutant I
C68S, C86S
A116C, A135C
(SEQ ID NO: 11) No change Variant J
C68S, C86S
A116C, I136C
(SEQ ID NO: 12) No change Mutant K
C68S, C86S
A74C
(SEQ ID NO: 13) change
(62 DEG C) Mutant L
C68S, C86S
L25C
(SEQ ID NO: 14) No change Mutant M
C68S, C86S
K137C
(SEQ ID NO: 15) No change Mutant N
C68S, C86S
K51C, A143C
(SEQ ID NO: 16) No change

Variant O
C68S, C86S
A74C, H49Y
(SEQ ID NO: 17) (65 DEG C)

Table 1 shows the degree of structural change of native bFGF and bFGF variants and the unfolded fraction of the temperature at 208 nm in circular dichroism analysis. 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.

In Table 1, SEQ ID NO: 1 is a mutant in which methionine is added as a starting sequence after removing two N-terminal amino acid sequences from the natural sequence.

As a result of measuring the Tm of the novel mutant produced in the above-mentioned invention, when compared with the results of the existing patent ((KR 10-2015-0078930 / PCT-KR2015-007734), it showed a Tm pattern similar to that of the existing HsbFGF, The sequence shows no significant effect on the structure of bFGF.

As shown in the existing patent, most of the structural changes showed the same structure as that of native bFGF, and the mutants added with disulfide bonds had no specific structure. In the case of Tm indicating heat stability, it was almost similar to the experiment of the existing patent. Almost all of them were the same as the bFGF mutant in comparison with the natural type bFGF at 58 ° C. Among the mutants, K7 ), The Tm value increased to 62 ° C, and the Tm indicating the thermal stability of mutant O (new HsbFGF) in which mutant K was substituted with tyrosine at 49th was increased to 65 ° C. Based on these experiments, it was confirmed that the thermal stability was improved and the data consistent with the contents of the existing patents were shown.

The bFGF, which showed good results through analysis of the structure and Tm using the solubility and circular dichroism among the wild type bFGF and mutants, was selected and cell proliferation assay was performed. Cell proliferation assays were performed with the NIH-3T3 cell line, a bFGF-sensitive skin cell. NIH-3T3 cells were maintained in DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. 2 × 10 ³ cells / well of NIH-3T3 cells were seeded on a 96-well culture plate. The NIH-3T3 cells cultured for 24 hours were treated with serum-free DMEM medium and then treated with the sample solution in DMEM medium containing 0.5% FBS for 72 hours after starvation. 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. 100 μl of DMSO was added to formazan crystal &Lt; / RTI > Absorbance was measured at 540 nm using a spectrophotometer. The susceptibility to the drug was compared with the percentage of the absorbance of the untreated well (control) in the drug-treated wells (Figs. 4-6, Fig. 8).

Experimental Example 3 Quantitative Analysis of Protein by Incubation of Natural and Mutant bFGF [

In order to confirm the stability of wild-type bFGF and its mutants, incubation at 37 ° C, 50 ° C and 60 ° C was carried out. In a phosphate buffer (PBS), which is most similar to the human body, native bFGF and its mutants were dissolved at 0.5 mg / ml and incubated in water bath at each temperature. The samples were sampled at 0, 24, 48 hours, 7 days, and 10 days and centrifuged at 13,000 rpm for 15 minutes at 4 ° C to quantitate proteins using nano drop. Over time, the concentrations of wild type bFGF and mutants were quantitated, and the wild type bFGF showed a more significant decrease than the mutant.

Based on the above results, incubation of existing HsbFGF, novel HsbFGF mutant and native bFGF with increased heat stability was proceeded. As a result, mutants were much more stable than native bFGF. Based on these experiments, the natural type, the existing HsbFGF and the new HsbFGF were incubated at 37 ° C for one month and the results were much better than those of the wild type.

EXPERIMENTAL EXAMPLE 4 SDS-PAGE and HPLC Analysis by Long-Term Incubation of Natural and Mutant bFGFs at 37 ° C [

In order to confirm the stability of wild-type bFGF, mutant K and novel HsbFGF mutants, incubation was carried out at 37 ° C for 100 days. Each of the wild-type bFGF and its mutants were dissolved in phosphate buffer solution at 0.5 mg / ml and incubated in a water bath at 37 ° C. The fractions were taken by date and centrifuged at 13,000 rpm for 15 minutes at 4 ° C to obtain supernatant, and proteins were analyzed by HPLC and SDS-PAGE.

As shown in Fig. 9, it was confirmed that (a) SDS-PAGE and (b) HPLC analysis of the new HsbFGF were preserved even after 100 days.

Mouse L929 cells (ECACC, no. 85011425) were maintained in DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. NIH-3T3 cells were seeded at 5 × 10 ³ cells / well in a 96-well culture plate. The sample solutions were treated with L929 cells for 24 hours, and cultured for 24 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. 100 μl of DMSO was added to formazan crystal &Lt; / RTI > Absorbance was measured at 540 nm using a spectrophotometer. The susceptibility to the drug was compared with the percentage of absorbance of the wells (control) not treated with the drug in the treated wells. As shown in Fig. 10, the newly produced HsbFGF was not found to be toxic.

Experimental Example 6: N-terminal analysis [

The purified protein was subjected to Edman N-terminal analysis method by Korea Institute of Mass Spectrometry. 0.5 mg / ml. Immerse the PVDF membrane in 100% MeOH for 30 seconds to 1 minute. Transfer is then carried out using a 0.45 μm PVDF membrane. Immerse the blotted membrane in the third distilled water for 5 minutes. Dyeing is carried out within 5 minutes using a dyeing reagent. After discarding the dyeing reagent, immediately discolor using a decoloring reagent. At the point where the protein band clearly appears, discard the decoloring reagent and wash it with the third distilled water. Air-dry the washed membrane thoroughly. (PTC) -polypetide, anilinothiazolamine (ATZ) -amino acid, and the final phenylthiohydantoin (PTH) derivative amino acid form stabilized by the initiation of the initial reaction in which N-terminal polypeptide chains are formed through phenylisothiocyanate And the sequence is sequentially confirmed by injecting the amino acid derivative produced in each repetition by HPLC. In the case of the PITC peak shown in FIG. 7, the remaining PITC reagent remained in the course of coupling the N-terminal amino acid with the PITC reagent, which is the peak shown in the mass analysis, which is not related to the N-terminal amino acid. Dptu in the following peaks is also a reagent used as a reference in chromatographic separation as a standard and standard reagent.

As shown in FIG. 7 (a), the mutant O (New HsbFGF) of the present invention was analyzed as a single component, whereas the Old HsbFGF of the conventional invention of FIG. 7 (b) And shows the superiority of the present invention.

&Lt; 110 > PnP Biopharm Co., Ltd. <120> A High-Stable Mutant of Basic Fibroblast Growth Factor with Modified N-Terminal Amino Acid Region, And Uses Thereof <130> P16-0253HS <160> 17 <170> Kopatentin 2.0 <210> 1 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 1 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 2 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 2 atgttgcccg aggatggcgg cagcggcgcc ttcccgcccg gccacttcaa ggaccccaag 60 cggctgtact gcaaaaacgg gggcttcttc ctgcgcatcc accccgacgg ccgagttgac 120 ggggtccggg agaagagcga ccctcacatc aagctacaac ttcaagcaga agagagagga 180 gttgtgtcta tcaaaggagt gtgtgctaac cgttacctgg ctatgaagga agatggaaga 240 ttactggctt ctaaatgtgt tacggatgag tgtttctttt ttgaacgatt ggaatctaat 300 aactacaata cttaccggtc aaggaaatac accagttggt atgtggcact gaagcgaact 360 gggcagtata aacttggatc caaaacagga cctgggcaga aagctatact ttttcttcca 420 atgtctgcta agagc 435 <210> 3 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 3 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Cys His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Cys Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 4 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 4 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Cys His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Cys Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 5 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 5 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Cys His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Cys Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 6 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 6 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Cys Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Cys Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 7 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 7 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Cys Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Cys Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 8 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 8 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 Cys Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 9 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 9 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Cys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Cys Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 10 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 10 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Cys Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Cys Arg Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 11 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 11 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Cys Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Cys Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 12 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 12 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Cys Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Cys Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 13 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 13 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Cys Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 14 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 14 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Cys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 15 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 15 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Cys Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145 <210> 16 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 16 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 His Ile Cys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Cys Lys 130 135 140 Ser 145 <210> 17 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 17 Met Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25 30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro 35 40 45 Tyr Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile 50 55 60 Lys Gly Val Ser Ala Asn Arg Tyr Leu Cys Met Lys Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr Ser 100 105 110 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145

Claims

Wherein at least two amino acids in the amino acid sequence of SEQ ID NO: 1 are replaced by serine and at least one amino acid is substituted by cysteine, wherein said mutant is substituted with serine at the 68th and 86th cysteines of SEQ ID NO: Stable basic fibroblast growth factor (bFGF) mutant in which the first alanine is substituted with cysteine.

The method of claim 1, wherein the mutant is a high-stability basic fibroblast growth cell in which the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, the 74th alanine is replaced with cysteine, and further the 49th histidine is substituted with tyrosine Factor (bFGF, basic fibroblast growth factor) mutant.

delete

A gene encoding a mutant of any one of claims 1 to 2.

delete

An expression vector comprising the gene of claim 5.

A transformant transformed by the expression vector of claim 7.

A method for producing high-stability basic fibroblast growth factor variant comprising the steps of:
(a) culturing the transformant of claim 8; And
(b) separating the mutant from the culture obtained in the step (a).

10. The method of claim 9, wherein step (b)
(c) disrupting the transformant and removing aggregates;
(d) separating and purifying the aggregate-removed supernatant using ion exchange resin chromatography; And
(e) separating and purifying the high-stability basic fibroblast growth factor variant after the ion-exchange resin using heparin affinity chromatography.

A cosmetic composition for improving skin condition comprising the high-stability basic fibroblast growth factor variant of any one of claims 1 to 2 as an active ingredient.

A pharmaceutical composition for preventing or treating skin diseases, which comprises the high-stability basic fibroblast growth factor variant of any one of claims 1 to 2 as an active ingredient.