KR101754272B1 - A human basic fibroblast factor-2 mutant with high stability and use of the same - Google Patents

Abstract

The present invention relates to high-stability basic fibroblast growth factor variants and uses thereof. More particularly, a high-stability basic fibroblast growth factor (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 . 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 human fibroblast growth factor-2 mutant having increased stability and a use thereof.

The present invention relates to high-stability basic fibroblast growth factor 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, bFGF (Basic Fibroblast Growth Factor, FGF-2) is composed of 154 amino acids and is composed of a polypeptide having a molecular weight of 17.123 Dalton. It plays an important role in development, angiogenesis and wound healing. FGF-2 is a potent mediator of wound healing, angiogenesis, and nervous system growth as mitogenic (mitogenic) and chemotactic factors.

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 disulfide bonds in its structure, There is a problem that it is greatly affected.

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

Thus, there is a need to develop novel bFGF variants that are more stable and active.

[Prior Patent Literature]

Korean Patent Publication No. 1020090083062

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 basic fibroblast growth factor (bFGF) mutant.

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.

Still another object of the present invention is to provide a method for producing a high-stability basic fibroblast growth factor (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 achieve the above object, the present invention provides a method for producing a high-stability basic fiber, wherein at least two amino acids in the amino acid sequence of SEQ ID NO: 1 are substituted with serine, one or more amino acids are substituted with cysteine, And provide basic fibroblast growth factor (bFGF) mutants.

The term "basic fibroblast growth factor (bFGF)" or "FGF-2 ", as used herein, refers to a basic protein with a molecular weight of about 18 kDa (pI 9.58), secreted predominantly in the pituitary gland and promoting the growth of various mesodermal 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 the method of protein molecule by computer, , Wherein the cysteine amino acid residue forming a disulfide bond with bFGF and another bFGF is substituted with a serine residue having a similar structure to increase stability against precipitation due to surface disulfide bond. In addition, stability is improved by reducing loop entropy by additionally producing a disulfide bond by replacing one residue near the loop in bFGF with cysteine. Also, it is characterized in that stability is improved by stabilizing the cavity structure of the protein by increasing the hydrogen bond and Van der walls interaction by substituting Ser with the Ala residue in bFGF.

According to a preferred embodiment of the present invention, the amino acid substituted with serine is the 69th cysteine and the 87th cysteine in the amino acid sequence of SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the cysteine-substituted amino acid is the 26th lysine in the amino acid sequence of SEQ ID NO: 1; The 34th isoleucine in the amino acid sequence of SEQ ID NO: 1; A 40th valine in the amino acid sequence of SEQ ID NO: 1; The 50th histidine in the amino acid sequence of SEQ ID NO: 1; The 52nd lysine in the amino acid sequence of SEQ ID NO: 1; The 75th alanine in the amino acid sequence of SEQ ID NO: 1; A 76th mesaionin in the amino acid sequence of SEQ ID NO: 1; The 117th alanine in the amino acid sequence of SEQ ID NO: 1; The 67th glycine in the amino acid sequence of SEQ ID NO: 1; The 68th valine in the amino acid sequence of SEQ ID NO: 1; The 70th alanine in the amino acid sequence of SEQ ID NO: 1; The 82nd leucine in the amino acid sequence of SEQ ID NO: 1; An 84th alanine in the amino acid sequence of SEQ ID NO: 1; The 108th serine in the amino acid sequence of SEQ ID NO: 1; The 136th alanine in the amino acid sequence of SEQ ID NO: 1; 137th isoleucine in the amino acid sequence of SEQ ID NO: 1; The 138th leucine in the amino acid sequence of SEQ ID NO: 1; And 144th alanine in the amino acid sequence of SEQ ID NO: 1, more preferably at least one selected from the group consisting of the 40th valine in the amino acid sequence of SEQ ID NO: 1; The 50th histidine in the amino acid sequence of SEQ ID NO: 1; The 52nd lysine in the amino acid sequence of SEQ ID NO: 1; The 75th alanine in the amino acid sequence of SEQ ID NO: 1; A 76th mesaionin in the amino acid sequence of SEQ ID NO: 1; The amino acid sequence of SEQ ID NO: 1, and the 117th amino acid sequence of SEQ ID NO: 1, and most preferably the 75th amino acid sequence of SEQ ID NO: 1.

As a further variant, a variant obtained by replacing the seventy-seventh residue of Ala, which is a residue exposed on the surface of the protein, with a serine is the most preferable variant.

That is, the bFGF mutant of the present invention is a bFGF mutant in which all of the 69th and 87th amino acid residues cysteine in the native human bFGF amino acid sequence (SEQ ID NO: 1) are substituted with serine, substituted with serine of 70th alanine, Alanine is further substituted by cysteine to form disulfide bonds in the molecule and the remaining amino acid sequence provides the same human bFGF mutein as the native amino acid sequence.

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 Examples 1 to 3, the bFGF mutant has an activity equivalent to that of the wild type, and the stability against heat is also remarkably increased. In the bFGF mutant, K75 of the present invention in which the amino acids 69 and 87 were substituted with serine and the amino acid 75 was substituted with cysteine and then the disulfide bond was induced was substituted with serine for the natural type bFGF, The bFGF mutant and the bFGF mutant in which the amino acid No. 75 was substituted with cysteine were improved in thermal stability. In addition, in the case of HsbFGF substituted with serine at the 70th alanine of K75, the thermal stability of the natural type and K75, which are the control group, was improved

According to another aspect of the present invention, the present invention provides a DNA sequence (e. G., 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 the bFGF gene is inserted into the pET21a vector was prepared and named "pSSB-bFGF" (disclosed in 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, .

The DNA encoding the bFGF mutant is a DNA encoding the amino acid of the substituted region of native bFGF. Preferred DNA sequences encoding bFGF variants are those in which the 69th and 87th codons are replaced with codons that encode serine and the 75th codon is replaced with a codon that codes for cysteine. In addition, in the case of HsbFGF substituted with serine at the 70th alanine of K75, the thermal stability of the natural type and K75, which are the control group, was improved

On the other hand, it is well known that the 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 codon degeneracy.

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 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 for non-glycosylated bFGF variants, and suitable vectors that can be used for expression in E. coli include, (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, there is provided a method of producing a high-stability basic fibroblast growth factor (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 transforming cell and separating the aggregate;

(d) removing the separated aggregates;

(e) separating and purifying the supernatant from which the aggregates have been removed by 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 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, bFGF variants expressed in transformed E. coli cells can be recovered from the cell culture or after extraction of the cells by suitable methods known to the proteochemical system.

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 separate the insoluble granular aggregates, and the separated aggregates are removed. The supernatant liquid from which the aggregates have been removed is separated and purified using ion exchange resin chromatography, and the ion-exchange resin is 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 closed windows include contusion or burys 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 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, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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 dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg.

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, surfactant- , 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 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 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 bFGF mutant of the present invention is excellent in heat stability and stability in an aqueous solution state, so that it is possible to produce functional cosmetics that do not lose activity unlike conventional natural bFGF products during distribution and storage, It is available.

Figure 1 shows an overview of the assembly of plasmid pSSB-bFGF.
Figure 2 shows SDS-PAGE results of native (bFGF) and bFGF mutants of the present invention after T cell (cell suspension after suspension) S (supernatant after cell disruption) P (insoluble aggregate after cell lysis).
FIG. 3 shows the difference in the Melting temperature (TM), which is an index of the thermal stability of the native bFGF and the bFGF mutant of the present invention. 3, the x-axis is temperature.
Fig. 4 shows the results of comparing the activity of the native bFGF with the bFGF mutant of the present invention.
FIG. 5 shows the stability comparison result after 20 days incubation at 25 ° C in the state of PBS (phosphate buffer saline) which is most similar to the state of the human bFGF mutant of native bFGF of the present invention.
FIG. 6 is an analysis using SDS-PAGE after final purification of native bFGF (A) and bFGF mutant (K75) (B) of the present invention.
7 shows the results of the difference between the native bFGF and the sbFGF mutant of the present invention and the TM (Melting temperature), an index of the thermal stability of HsbFGF. In FIG. 7, the x-axis is the temperature.
Figure 8 shows the stability comparison results after one week incubation at 50 ° C in PBS (Phosphate buffer saline) conditions most similar to the sbFGF mutant and HsbFGF human body of the present invention.
FIG. 9 shows the stability comparison result after incubation at 60 ° C for 5 days in a state of PBS (phosphate buffer saline) which is most similar to the state of native bFGF, sbFGF mutant of the present invention and HsbFGF human body.
FIG. 10 shows HPLC quantitative comparison results after incubation at 60 ° C for 5 days in the state of PBS (phosphate buffer saline) most similar to that of natural bFGF, sbFGF mutant of the present invention and HsbFGF human body. 10, the x-axis is time (min), the Y-axis is mAU
Fig. 11 shows the results of bFGF activity comparison between wild-type bFGF and bFGF mutants of the present invention.

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 present invention as defined by the appended claims. It will be obvious to you.

Manufacturing example

DNA  Construction ( 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 ™ HF 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 Protein expression )

The expression vector IPTG (isopropyl-1-thio-β-D-galactopyranoside) and antibiotics ampicillin and chloramphenicol were purchased from Sigma. E. coli culture Bacto tryptone and yeast extract used in the preparation of LB medium were purchased from BD (Becton Dicknson), and NaCl was purchased from Duksan.

Protein purification ( Protein 정화 )

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  modelling( Homology modeling )

Homology modeling was done by Modeller (Andrej Sali).

Energy Minimization Energy minimization )

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

Disulfide bond  prediction( Disulfide predict )

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

Bond  Distance measurement

Protein contact map visualization (Andreas Viklund.) Was used for plotting progrea to measure disulfide bond distance.

Structure of protein

4FGF and 1BLA 1BLD registered in the PDB were used.

Vector system ( Vector system )

PET21a vector (Novagen) was used as an expression vector for producing mutant bFGF. The wild-type bFGF gene was obtained from PnP biopharm, and amplified by polymerase chain reaction (PCR) 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 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, and a quikchange mutagenesis method using pfu Ultra ™ DNA polymerase using a primer . The Dpn I reaction was performed to remove the native bFGF template, and the resultant was transformed into Top 10, and the mutant was confirmed by sequencing.

Natural type  And Mutant bFGF Manifestation of

The bFGF-inserted recombinant vector was transformed into E. coli BL21 (DE3) by Heat shock method. This E. coli strain was inoculated into 500 ml of LB medium containing 50 μg / ml ampicillin and grown at 37 ° C until the O.D 600 value reached 0.6. Then, 0.5 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added and incubated for 4 hours. When O.D 600 value became 2.0 or more, cells were centrifuged at 8000 rpm for 10 minutes.

Cell disruption

Cells were disrupted to obtain proteins in E. coli expressing bFGF protein. The harvest cells were suspended in 20 mM sodium phosphate buffer (pH 7.0) and disrupted with a sonicator at 4 ° C. Subsequently, the mixture was centrifuged at 13000 rpm for 15 minutes at 4 ° C to remove the inclusion body, and then the supernatant was collected and confirmed by SDS-PAGE.

Transformants ( transformation ) Tablets

The sonicated cell lysate was centrifuged at 13000 rpm for 15 minutes at 4 ° C. The supernatant was collected and filtered through a 0.45 μm filter. The solution was purified by FPLC (Fast Performance Liquid chromatography) SP column and Heparin column. The purification conditions were as follows: 1 mM NaCl B in 20 mM sodium phosphate (pH 7.0) buffer, 100 mM NaCl solution A and 20 mM sodium phosphate (pH 7.0) buffer were added to the SP column at a rate of 2 ml / And the fractions containing the bFGF protein of about 18 KDa size were collected. Then, 2M NaCl B was added to a heparin affinity column in a buffer of 20 mM sodium phosphate (pH 7.0) and a buffer solution of 20 mM sodium phosphate (pH 7.0) in a linear gradient from 0% A to 100% B at a rate of 2 ml / The fraction containing the bFGF protein of about 18 KDa in size was collected after being eluted with a gradient. At this time, fractions containing bFGF were confirmed by SDS-PAGE analysis and then quantified.

Molecular Modeling Molecular modeling )

A candidate disulfide bondable group 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. We then used the Yasara energy minimization server to analyze the formation of disulfide bonds and proceed with energy minimization using AMBER force filed FF99 of chimera. The structure of the resulting protein was aligned with the native bFGF to determine the RMSD value of 0.5 or less.

CD( Circular dichroism )

For the structural analysis and TM measurement of wild-type bFGF and mutants, bFGF is dissolved in 20 mM sodium phosphate (pH 7.05), and the final concentration is adjusted to 0.2 mg / ml. The cell was immersed in a 0.1 cm cell and the band width 1 nm, response 0.25 sec, data pitch 0.1 nm, scanning speed 20 nm / min, cell length 1 cm, accumulation 8 times, temperature 20 ° C. Respectively. Melting temperature was 0.2 mg / ml in 0.1 cm cell 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.

Residue numbers and predicted results for disulfide bond are shown in Table 1.

Cell proliferation assay ( Cell proliferation assay )

Experiments using cell proliferative capacity were carried out with GENEWELL Co., Ltd. to confirm that the produced wild type bFGF and the mutant actually showed activity. 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 in a 96-well culture plate. NIH-3T3 cells cultured for 24 hours were starvated in serum-free DMEM medium and then treated with 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. 100 μl of DMSO was added to formazan crystal ≪ / 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 )

Incubation tests of wild type bFGF and mutants were performed to confirm the storage at room temperature. Each wild-type FGF-2 and mutants were dissolved in 1X PBS (pH 7.3) at 0.5 mg / ml and incubated at 37 ° C, 50 ° C and 60 ° C in a water bath. The supernatant was obtained by centrifugation at 4 ° C for 15 minutes at 13000 rpm by sampling on a 24-hour basis. Nano-drop determination and HPLC analysis were performed.

< Example  1: People bFGF cDNA Containing pSSB - bFGF  Construction of Plasmids>

A DNA encoding bFGF was prepared by polymerase chain reaction using a human monocyte cDNA library as a template and a primer. The nucleotide sequence of the primers used was as follows: sense primer 5'-GGCGGGCATATGCCCGCCTTGCCCGAGG-3 '(SEQ ID NO: 3) and antisense primer 3'-TGATGAGGATCCTCATCAGCTCTTAGCAGACAT-5' (SEQ ID NO: 4).

The bFGF portion of FIG. 1B was amplified using the primers described above. 1 μg of the amplified DNA fragment was dissolved in 50 μl of TE (pH 8.0) solution and then 2 units of Nde I (NEB) and 2 units of Bam HI (NEB), and reacted at 37 ° C for 2 hours to have a Nde I restriction site at the 5'-end and a Bam HI restriction enzyme site at the 3'-end. 20 ng of this DNA fragment was treated with Nde I and Bam HI in the same manner and then treated with 20 ng of pET21a (+) plasmid (Novagen) in a DNA purification kit (GeneAll) Mu] l of TE (pH 8.0) solution, followed by addition of 1 unit of T4 DNA ligase (NEB), followed by reaction at 16 [deg.] C for 4 hours. The resulting plasmid was named pSSB-bFGF.

< Example  2: People bFGF Escherichia coli Divertic  Manufacturing>

The expression plasmid pSSB-bFGF 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. After incubation at 37 ° C for 12 hours, 100% glycerol and 1: 1 were mixed and stored at -70 ° C.

< Example  3: People bFGF Purification of>

The stalk prepared in Example 2 was inoculated into 10 ml of LB medium (LB / ampicillin) and cultured for 12 hours or more. Then, it was 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 an absorbance of OD of 0.4-0.5 at 600 nm. The cells were incubated at 37 ° C for 4 hours with shaking at 200 rpm and then centrifuged at 8000 rpm for 10 minutes to obtain pellet of Escherichia coli. The pellet was suspended in 25 ml of 20 mM sodium phosphate (pH 7.0) buffer, and the cells were disrupted by ultrasonication.

The sonicated cell lysate was centrifuged at 13000 rpm for 15 minutes at 4 ° C. The supernatant was filtered through a 0.45 μm filter and purified by FPLC (Fast Performance Liquid chromatography), SP column and Heparin column. For purification, 20 mM sodium phosphate (pH 7.0) buffer solution, 100 mM NaCl solution A, and 2 mM NaCl B solution in 20 mM sodium phosphate (pH 7.0) buffer were added to the SP column at a rate of 2 ml / The fraction containing the bFGF protein of about 18 KDa was collected after elution with a linear gradient to% B. Then, 2 M NaCl B was added to 20 mM sodium phosphate (pH 7.0) buffer in 100 mM NaCl solution A and 20 mM sodium phosphate (pH 7.0) buffer to a linear gradient from 50% A to 100% B at a rate of 2 ml / And the fractions containing the bFGF protein of about 18 KDa size were collected. At this time, the fractions containing bFGF were confirmed by SDS-PAGE analysis, followed by quantification and obtaining 10 mg of bFGF

< Example  4: pSSB - bFGF Mutant  Construction of Plasmids>

PSSB-bFGF mutant plasmids were prepared by PCR using pfuUltra ™ HF DNA polymerase as the template for the native pSSB-bFGF plasmid using two complementary primers corresponding to each variant. After digesting the native pSSB-bFGF plasmid using Dpn I, the plasmid was 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-bFGF mutant plasmids. The base sequence of the primers used was as follows:

(SEQ ID NO: 5) and antisense primer 3'-CAG GTA ACG GTT (SEQ ID NO: 5) at the time of substitution with serine codon TCT at the 69th cysteine codon TTT was substituted with TCT ATT AAA GGA GTG TCT GCT AAC CGT TAC CTG- AGC AGA CAC TCC TTT GAT AGA-5 '(SEQ ID NO: 6);

(SEQ ID NO: 7) and the antisense primer 3'-ACA CTC ATC CGT (SEQ ID NO: 7) at the time of substitution with TCT, which is a codon of serine at codon 87 of cysteine, the sense primer 5'-TTA CTG GCT TCT AAA TCT GTT ACT GAG GAG TGT- AAC AGA TTT AGA AGC CAG TAA-5 '(SEQ ID NO: 8);

GCT AAC CGT TAC CTG TGC ATG AAG GAA GAT GGA-3 '(SEQ ID NO: 9) and antisense primer 3'-TCC ATC TTC CTT (SEQ ID NO: 9) when GCT, the codon of the 75th alanine, CAT GCA CAG GTA ACG GTT AGC-5 '(SEQ ID NO: 10);

AAG CGG CTG TAC TGC TGC AAC GGG GGC TTC TTC-3 '(SEQ ID NO: 11) and antisense primer 3'-GAA GAA GCC CCC at the time of substitution of the codon of the 26th lysine with the codon of cysteine TGC GTT GCA GCA GTA CAG CCG CTT-5 '(SEQ ID NO: 12);

(SEQ ID NO: 13) and the antisense primer 3'-TCG GCC GTC (SEQ ID NO: 13), the sense primer 5'-GGC TTC TTC CTG CGC TGC CAC CCC GAC GGC CGA- GGG GTG GCA GCG CAG GAA GAA GCC-5 '(SEQ ID NO: 14);

 CTC CCG GAC CCC (SEQ ID NO: 15) and antisense primer 3'-CTC CCG GCC CCC GAC GGC CGA TGC GAC GGG GTC CGG GAG-3 '(SEQ ID NO: 15) at the time of substitution of the 40th valine codon GTG with the codon TGC of cysteine. GTC GCA TCG GCC GTC GGG GTG-5 '(SEQ ID NO: 16);

GAG AAG AGC GAC CCT TGC ATC AAG CTA CAA CTT-3 '(SEQ ID NO: 17) and the antisense primer 3'-AAG TTG TAG CTT GAT GCA AGG GTC GCT CTT CTC-5 '(SEQ ID NO: 18);

AGC GAC CCT CAC ATC TGC CTA CAA CTT CAA GCA-3 '(SEQ ID NO: 19) and antisense primer 3'-TGC TTG AAG TTG TAG GCA GAT GTG AGG GTC GCT-5 '(SEQ ID NO: 20);

(SEQ ID NO: 21) and the antisense primer 3'-TCT TCC (SEQ ID NO: 21) and the sense primer 5'-AAC CGT TAC CTG GCT TGC AAG GAA GAT GGA AGA- ATC TTC CTT GCA AGC CAG GTA ACG GTT-5 '(SEQ ID NO: 22);

ACC AGT TGG TAT GTG TGC CTG AAG CGA ACT GGG-3 '(SEQ ID NO: 23) and antisense primer 3'-CCC AGT TCG CTT (SEQ ID NO: 23) at the time of substitution of the 117th alanine codon GCA with the codon of cysteine TGC CAG GCA CAC ATA CCA ACT GGT-5 '(SEQ ID NO: 24);

(SEQ ID NO: 25) and the antisense primer 3'-ACG GTT AGC AGA (SEQ ID NO: 25) and the antisense primer 3'-ACG GTT AGC AGA CAC GCA TTT GAT AGA CAC AAC-5 '(SEQ ID NO: 26);

GTG TCT ATT AAA GGA TGC TCT GCT AAC CGT TAC-3 '(SEQ ID NO: 27) and antisense primer 3'-GTA ACG GTT AGC AGA GCA TCC TTT GAT AGA CAC-5 '(SEQ ID NO: 28);

ATC AAA GGA GTG TCT TGC AAC CGT TAC CTG GCT-3 '(SEQ ID NO: 29) and antisense primer 3'-AGC CAG GTA ACG (SEQ ID NO: 29) when GCT, the 70th alanine codon, was substituted with TGC, GTT GCA AGA CAC TCC TTT GAT-5 '(SEQ ID NO: 30);

(SEQ ID NO: 31) and the antisense primer 3'-AGA TTT AGA AGC (SEQ ID NO: 31) and the antisense primer 3'-AGA TTT AGA AGC at the substitution of the 82nd lucine codon TTA with the cysteine codon TGC. CAG GCA TCT TCC ATC TTC CTT-5 '(SEQ ID NO: 32);

GAT GGA AGA TTA CTG TGC TCT AAA TCT GTT ACG-3 '(SEQ ID NO: 33) and antisense primer 3'-CGT AAC AGA TTT (SEQ ID NO: 33) when GCT, the codon of 84th alanine, AGA GCA CAG TAA TCT TCC ATC-5 '(SEQ ID NO: 34);

3 '(SEQ ID NO: 35) and the antisense primer 3'-ACT GGT GTA TTT (SEQ ID NO: 35) at the time of substitution with the codon TGC of the cysteine codon of the 108th serine, 5'-TAC AAT ACT TAC CGG TGC AGG AAA TAC ACC AGT- CCT GCA CCG GTA AGT ATT GTA-5 '(SEQ ID NO: 36);

3 '(SEQ ID NO: 37) and antisense primer 3'-TGG AAG AAA AAG (SEQ ID NO: 37) were used in place of the sense primer 5'-GGA CCT GGG CAG AAA TGC ATA CTT TTT CTT CCT- TAT GCA TTT CTG CCC AGG TCC-5 '(SEQ ID NO: 38);

ATT-3 '(SEQ ID NO: 39) and antisense primer 3'-CAT TGG AAG (SEQ ID NO: 39) were used as the sense primer 5'-CCT GGG CAG AAA GCT TGC CTT TTT CTT CCT ATG- AAA AAG GCA AGC TTT CTG CCC AGG-5 '(SEQ ID NO: 40);

(SEQ ID NO: 41) and the antisense primer 3'-AGA CAT TGG AAG (SEQ ID NO: 41) at the substitution of the 138th codon of the lysine with the codon TGC of cysteine, the sense primer 5'-GGG CAG AAA GCT ATA TGC TTT CTT CCA ATG TCT- AAA GCA TAT AGC TTT CTG CCC-5 '(SEQ ID NO: 42); And

TCT CCA ATG TCT TGC AAG AGC TGA TGA-3 '(SEQ ID NO: 43) and antisense primer 3'-TCA TCA GCT CTT GCA when the GCT, the 144th alanine codon, was substituted with TGC, the codon of cysteine AGA CAT TGG AAG AAA-5 '(SEQ ID NO: 44).

(SEQ ID NO: 45) and the antisense primer 3'-CAG GTA ACG GTT AGA AGA (SEQ ID NO: 45) and the antisense primer 3'-CAG at the substitution of the 70th alanine codon GCT with the serine codon TGt. CAC TCC TTT -5 '(SEQ ID NO: 46).

< Example  5: bFGF Mutant  Production and refining>

E. coli BL21 (DE3) was transformed into each of the expression plasmids of the bFGF mutant of Example 4 in the same manner as in Example 2, and the cells were cultured in 500 ml of LB medium (LB / ampicillin) To obtain bFGF of about 18 kDa in size. The amount of mutant obtained was variable according to the mutant, and about 4 ~ 12 mg of bFGF was obtained depending on the mutant. The purity was 98% or more.

Each of the bFGF variants is as follows:

A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 34th isoleucine and the 67th glycine are substituted with cysteine;

A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 34th isoleucine and the 70th alanine are substituted with cysteine;

Mutants in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 34th isoleucine and 84th alanine are substituted with cysteine;

A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 40th valine and 82nd leucine are replaced by cysteine;

Mutants in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine and the 40th valine and 84th alanine are substituted with cysteine;

Mutants in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 50th histidine and 69th cysteine are replaced by cysteine;

A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 52nd lysine and the 68th valine are substituted with cysteine;

A mutant wherein the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 76th methionine and 108th serine are substituted with cysteine;

A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 117th alanine and 136th alanine are substituted with cysteine;

A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 117th alanine and 137th isoleucine are substituted with cysteine;

Mutants in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine and the 75th alanine is substituted with cysteine;

A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 26th lysine and 87th cysteine are substituted with cysteine;

A mutant wherein the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine and the 138th leucine is replaced with cysteine;

Mutants in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 52nd lysine and 144th alanine are substituted with cysteine; And

The 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 75th alanine is substituted with cysteine, A mutant in which the 70th alanine is substituted with serine.

The purified form of the native form and the variant can be purified through SP-sephrose and haparin affinity column. Both species eluted at about 400 mM NaCl in the SP column and 1.5M NaCl in the heparin column. After final heparin affinity column purification, SDS PAGE analysis was performed.

As shown in FIG. 6, dimer and trimer were observed in the case of the natural type, while it was confirmed that the variant existed in the form of a single band in the monomer size. It can be seen that the dimer and trimmer with no activity are completely removed and exist in the monomer state.

Figure 6 shows SDS-PAGE results of the native (A) and variant (B) after final purification.

< Experimental Example  One: Natural type Mutant bFGF of circular dichroism Structural analysis using>

The structure and thermal stability of the purified bFGF variants of Example 5 were measured by circular dichroism analysis using a J-810 spectrometer (JASCO). The native bFGF used in Example 3 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.

In order to analyze the thermal stability, Tm (melting temperature) 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 a rate of 1 占 폚 / min from 20 占 폚 to 95 占 폚. The results are shown in Table 2.

Variant bFGF designation Structural change (Tm) Variant bFGF designation Structural change (Tm) Variant bFGF designation Structural change (Tm) Natural type bFGF
(SEQ ID NO: 1) -
(57.5 DEG C) Mutant
A34-67 decrease
48 ° C Mutant
B34-70 No change Mutant
C34-84 No change Mutant
D40-82 No change Mutant
E40-84 No change Mutant
F50-69 No change Mutant
G52-68 No change Mutant
H76-108 No change Mutant
I117-136 No change Mutant
J117-137 No change Mutant
K75 change
(62 DEG C) Mutant
L26-87 No change Mutant
M138 No change Mutant
N52-144 No change

Mutant
A70S (65 DEG C)

Table 2 shows the results of measurement of the degree of structural change for the native bFGF and bFGF mutants and the fraction unfolded at the wavelength of 208 nm in the circular dichroism 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 value is analyzed by measuring the melting temperature within the range of 20 to 95 ° C.

The bFGF mutants are mutants in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and further, the residues at respective positions are replaced with cysteines to induce intramolecular disulfide bonds.

As a result, most of the structural changes showed the same structure as that of native bFGF and there was no change, and the mutants added with disulfide bond had no specific structure. The thermal stability of the mutant bFGF was almost the same as that of the native bFGF at 58 ° C, and the heat stability was improved up to 62 ° C in the K75 mutant. This means that the thermal stability is increased by artificially adding disulfide bonds by replacing one amino acid with cysteine.

In order to confirm the conspicuousness of the K75 mutant in which the 69th and 87th cysteines of SEQ ID NO: 1, which are specific positions of the present invention, are substituted with serine and the 75th alanine is further substituted with cysteine, the natural bFGF , A bFGF mutant (Cys? Ser variant) in which only the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, a bFGF mutant (natural + disulfide bond) in which only the 75th alanine of SEQ ID NO: The mutant (Cys? Ser mutant + disulfide bond, the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine, and the 75th alanine was replaced with cysteine).

As a result, as shown in Fig. 3, the TM of natural type bFGF was about 57.5 DEG C, the TM of Cys-Ser mutant was 58 DEG C, the TM of natural type + disulfide bond was 61.5 DEG C, the K75 mutant (Cys? Ser mutant + disulfide bond TM was confirmed at 62 ° C, which means that the thermodynamic stability of the K75 mutant was increased.

In a further experiment, the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine, and the Tm indicating the thermal stability of HsbFGF substituted with serine at the 70th alanine in the K75 mutant with the 75th alanine as the cysteine was 58 DEG C It was confirmed that the thermal stability was improved up to 65 ° C as compared with the wild type bFGF and the 62 ° C K75 mutant. This means that one amino acid on the surface is substituted with serine to stabilize the cavity inside the protein and the thermal stability due to the newly formed hydrogen bond and van deer waals interaction is increased.

As a result, as shown in Fig. 7, the TM of natural bFGF was found to be about 57.5 DEG C, the TM of K75 mutant (Cys? Ser variant + disulfide bond) was found to be 62 DEG C and HsbFGF (K75 + Ala? Ser) Which means that the thermodynamic stability of the mutant is increased at 65 ° C.

< Experimental Example  2: Natural type Mutant bFGF Cell proliferation assay>

The bFGF and bFGF mutants were analyzed by solubility and circular dichroism, and their results were analyzed. Cell proliferation assays were performed with NIH3T3 cell line, a skin cell sensitive to bFGF, with the recommendation of GENE Well. 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 &gt; 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 FIGS. 4 and 10, the bFGF mutant exhibits cell proliferation similar to that of native bFGF.

< Experimental Example  3: Natural type Mutant bFGF of incubation Quantitative analysis of proteins according to>

Natural bFGF and mutants [bFGF mutant (Cys? Ser mutant) in which only the 69th and 87th cysteine of SEQ ID NO: 1 was substituted with serine, bFGF mutant in which only the 75th alanine of SEQ ID NO: 1 was substituted with cysteine + disulfide bond) and the K75 mutant (Cys? Ser mutant + disulfide bond, the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine and the 75th alanine was replaced with cysteine) , And a short-term incubation test was performed at 37 ° C. In the case of PBS (phosphate buffer saline), which is most similar to the human body, native bFGF and its mutants were dissolved at 0.5 mg / ml and incubated in a water bath at 37 ° C. 48 hours, 7 days, and 10 days, and centrifuged at 13000 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. The results are shown in Table 3.

bFGF name Day 0 After 48h After one week After 10days Natural type 100 56 26 16 CYS → SER mutant 100 88 80 62 Natural form + disulfide 100 92 78 64 K75 (CYS → SER mutant + disulfide bond) 100 90 88 80

Based on the above results, it was confirmed whether the K75 mutant had long-term storage stability in comparison with the native bFGF in the PBS (phosphate buffer saline) state most similar to the human body using the heat-stable K75 mutant. First, native bFGF and K75 mutants were dissolved in phosphate buffer saline (PBS) at the same concentration, followed by incubation at 25 ° C for 20 days. After centrifugation, the supernatant was quantitatively analyzed using HPLC.

As a result, as shown in FIG. 5, the K75 mutant in the incubation was much more stable than the native bFGF, and this shows the superiority of the K75 mutant of the present invention.

< Experimental Example  4: Natural type Mutant bFGF 50,60? incubation In accordance HPLC  Analysis>

(BFGF variant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine and the 75th alanine is substituted with cysteine) and the HsbFGF K75 mutant (Cys? Ser Mutant + disulfide bond, a bFGF mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 75th alanine is substituted with cysteine, and the 70th alanine is substituted with serine) Incubation test was carried out for 5 days at 60 ℃ for one week. Each native bFGF and its mutants were dissolved in PBS (phosphate buffer saline) at 0.5 mg / ml and incubated in a water bath at 50 ° C and 60 ° C. The samples were centrifuged at 13000 rpm for 15 minutes at 4 ° C, and the supernatant was collected and analyzed by HPLC and UV spectrometer.

As a result, as shown in Fig. 8, in the quantification using the UV spectrometer, the native bFGF could not be quantified after 5 days. In the case of the K75 mutant, 38% remained after 7 days, and 72% in the case of hsbFGF.

In addition, as shown in FIG. 9, in the quantification using a 60 ° C UV spectrometer, almost no bFGF was detected from day 3 after the third day, whereas only 22% remained after 5 days for K75 and 40% after 5 days for HsbFGF I could see that

In the analysis of HPLC using the results shown in FIG. 10, the native bFGF could not be quantified by HPLC after 7 days. In the case of K75, more than 60% of 30% HsbFGF remained.

&Lt; 110 > PnP Biopharm Co., Ltd. <120> A human basic fibroblast factor-2 mutant with high stability and          use of the same <130> HY150306 <160> 46 <170> Kopatentin 2.0 <210> 1 <211> 146 <212> PRT <213> Homo sapiens <400> 1 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His   1 5 10 15 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu              20 25 30 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp          35 40 45 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser      50 55 60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly  65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu                  85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr             100 105 110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser         115 120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala     130 135 140 Lys Ser 145 <210> 2 <211> 438 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 2 cccgccttgc ccgaggatgg cggcagcggc gccttcccgc ccggccactt caaggacccc 60 aagcggctgt actgcaaaaa cgggggcttc ttcctgcgca tccaccga cggccgagtt 120 gacggggtcc gggagaagag cgaccctcac atcaagctac aacttcaagc agaagagaga 180 ggagttgtgt ctatcaaagg agtgtctagc aaccgttacc tgtgcatgaa ggaagatgga 240 agattactgg cttctaaatc tgttacggat gagtgtttct tttttgaacg attggaatct 300 aataactaca atacttaccg gtcaaggaaa tacaccagtt ggtatgtggc actgaagcga 360 actgggcagt ataaacttgg atccaaaaca ggacctgggc agaaagctat actttttctt 420 ccaatgtctg ctaagagc 438 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggcgggcata tgcccgcctt gcccgagg 28 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgatgaggat cctcatcagc tcttagcaga cat 33 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tctatcaaag gagtgtctgc taaccgttac ctg 33 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 caggtaacgg ttagcagaca ctcctttgat aga 33 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ttactggctt ctaaatctgt tacggatgag tgt 33 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 acactcatcc gtaacagatt tagaagccag taa 33 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gctaaccgtt acctgtgcat gaaggaagat gga 33 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tccatcttcc ttcatgcaca ggtaacggtt agc 33 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aagcggctgt actgctgcaa cgggggcttc ttc 33 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gaagaagccc ccgttgcagc agtacagccg ctt 33 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggcttcttcc tgcgctgcca ccccgacggc cga 33 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tcggccgtcg gggtggcagc gcaggaagaa gcc 33 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 caccccgacg gccgatgcga cggggtccgg gag 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ctcccggacc ccgtcgcatc ggccgtcggg gtg 33 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gagaagagcg acccttgcat caagctacaa ctt 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 aagttgtagc ttgatgcaag ggtcgctctt ctc 33 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 agcgaccctc acatctgcct acaacttcaa gca 33 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 tgcttgaagt tgtaggcaga tgtgagggtc gct 33 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 aaccgttacc tggcttgcaa ggaagatgga aga 33 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tcttccatct tccttgcaag ccaggtaacg gtt 33 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 accagttggt atgtgtgcct gaagcgaact ggg 33 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 cccagttcgc ttcaggcaca cataccaact ggt 33 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 gttgtgtcta tcaaatgcgt gtctgctaac cgt 33 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 acggttagca gacacgcatt tgatagacac aac 33 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gtgtctatca aaggatgctc tgctaaccgt tac 33 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gtaacggtta gcagagcatc ctttgataga cac 33 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 atcaaaggag tgtcttgcaa ccgttacctg gct 33 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 agccaggtaa cggttgcaag acactccttt gat 33 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 aaggaagatg gaagatgcct ggcttctaaa tct 33 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 agatttagaa gccaggcatc ttccatcttc ctt 33 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 gatggaagat tactgtgctc taaatctgtt acg 33 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 cgtaacagat ttagagcaca gtaatcttcc atc 33 <210> 35 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 tacaatactt accggtgcag gaaatacacc agt 33 <210> 36 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 actggtgtat ttcctgcacc ggtaagtatt gta 33 <210> 37 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 ggacctgggc agaaatgcat actttttctt cca 33 <210> 38 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 tggaagaaaa agtatgcatt tctgcccagg tcc 33 <210> 39 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 cctgggcaga aagcttgcct ttttcttcca atg 33 <210> 40 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 cattggaaga aaaaggcaag ctttctgccc agg 33 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 gggcagaaag ctatatgctt tcttccaatg tct 33 <210> 42 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 agacattgga agaaagcata tagctttctg ccc 33 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 tttcttccaa tgtcttgcaa gagctgatga 30 <210> 44 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 tcatcagctc ttgcaagaca ttggaagaaa 30 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 aaaggagtgt cttctaaccg ttacctg 27 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 caggtaacgg ttagaagaca ctccttt 27

Claims (12)

Stable basic fibroblast growth factor (FBS) in which the 69th and 87th cysteines in the amino acid sequence of SEQ ID NO: 1 are substituted with serine, the 75th alanine is further substituted with cysteine, and the 70th alanine is substituted with serine, bFGF) mutant. delete delete delete A gene encoding the mutant of claim 1. 6. The gene according to claim 5, wherein the gene consists of the DNA sequence of SEQ ID NO: 2. An expression vector comprising the DNA sequence of claim 5. A transformant transformed by the expression vector of claim 6. A method for producing a high-stability basic fibroblast growth factor (bFGF) mutant 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 transforming cell and separating the aggregate;
(d) removing the separated aggregates;
(e) separating and purifying the supernatant from which the aggregates have been removed by 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. A cosmetic composition for improving skin condition comprising the high-stability bFGF mutant of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating skin diseases comprising the high-stability bFGF mutant of claim 1 as an active ingredient.

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