Human fibroblast growth factor-2 mutant with increased stability, and use thereof
11207257 · 2021-12-28
Assignee
Inventors
Cpc classification
A61K8/64
HUMAN NECESSITIES
International classification
Abstract
The present disclosure relates to a highly stable basic fibroblast growth factor mutant, and a use thereof. More specifically, the present disclosure provides: a highly stable basic fibroblast growth factor (bFGF) mutant, in which two or more amino acids in an amino acid sequence of SEQ ID NO: 1 are substituted with serine and one or more amino acids are substituted with cysteine; a DNA base sequence encoding the bFGF mutant; an expression vector including the DNA base sequence; a transformant transformed by the expression vector; a method of producing the bFGF mutant; and a composition including the bFGF mutant as an active ingredient. According to the present disclosure, the bFGF mutant of the present disclosure has excellent stability in an aqueous solution state and excellent thermal stability, and thus it is possible to produce functional cosmetics and skin inflammation medicines which do not lose activity, unlike conventional wild-type bFGF products, even during distribution and storage.
Claims
1. A basic fibroblast growth factor (bFGF) mutant, wherein the mutant is a bFGF mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, the 75th alanine of SEQ ID NO: 1 is substituted with cysteine, and the 50th histidine of SEQ ID NO: 1 is substituted with tyrosine.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
(12) Hereinafter, it will be apparent to a person having ordinary skill in the technical field to which the present disclosure pertains that the examples are for illustrative purposes only in more details and that the scope of the present disclosure is not construed as being limited by these examples without departing from gist of the present disclosure.
(13) Experimental Methods and Materials
(14) DNA Construction
(15) The protein expression vector pET21a (
(16) Protein Expression
(17) The expression vector IPTG (isopropyl-1-thio-β-D-galactopyranoside) and antibiotics ampicillin and chloramphenicol were both purchased from Sigma. Bacto tryptone and yeast extract used in the preparation of E. coli culture LB medium were purchased from BD (Becton Dicknson), and NaCl was purchased from Duksan.
(18) Protein Purification
(19) 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 GE healthcare's SP-sephrose, heparin affinity column.
(20) FPLC
(21) FPLC used GE UPC-800.
(22) CD (Circular Dichroism)
(23) The J-810 spectropolarimeter from Jasco was used for the CD.
(24) Homology Modeling
(25) Homology modeling used Modeller (Andrej Sali lab).
(26) Energy Minimization
(27) Energy minimization used Amber 99FF force filed included in Chimera.
(28) Disulfide Prediction
(29) YASARA Web server was used to predict the disulfide bond formation.
(30) Disulfide Bond Distance Measurement
(31) As a plotting program that measures the distance that enables disulfide bonds, protein contact map visualization (Andreas Viklund.) was used.
(32) Structure of Protein
(33) 4FGF and 1BLA 1BLD registered in the PDB were used.
(34) Vector System
(35) pET21a vector (Novagen) was used as an expression vector for producing mutant bFGF. The wild-type bFGF gene was obtained from PnP biopharm Co., Ltd., and the wild-type was amplified by PCR (polymerase chain reaction) using the following primers. The PCR products thus obtained were treated with Nde I and Xho I restriction enzymes and inserted into pET21a vector, and then bonded.
(36) Point Mutation
(37) In order to increase the stability of bFGF, the amino acid portion 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 the following primer (following Example 4) was used for amplification. To remove the wild-type bFGF template used, Dpn I reaction was performed to transform Top10, and mutants were identified by sequencing.
(38) Expression of Wild-Type and Mutant bFGF
(39) The bFGF-inserted recombinant vector was transformed into E. coli BL21 (DE3) by Heat shock method. The E. coli strain was inoculated into 500 ml LB medium containing 50 μg/ml ampicillin and grown at 37° C. until the O.D600 value reached 0.6. Then, 0.5 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added and cultured for 4 hours. When O. D600 value was 2.0 or more, cells were centrifuged at 8000 rpm for 10 minutes.
(40) Cell Disruption
(41) 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. Thereafter, the insoluble material (inclusion body) was removed by centrifugation at 13000 rpm for 15 minutes at 4° C., and the supernatant was selected and confirmed by SDS-PAGE.
(42) Purification of Transformants
(43) The cell solution disrupted by sonication was centrifuged at 13000 rpm for 15 minutes at 4° C. The supernatant was harvested, filtered through a 0.45 μm filter, and purified by FPLC (Fast Performance Liquid chromatography) SP column and Heparin column. The purification conditions were as follows: 100 mM NaCl solution A in 20 mM sodium phosphate (pH 7.0) buffer solution and 1M NaCl B in 20 mM sodium phosphate (pH 7.0) buffer solution were spilled to elute in a linear gradient from 0% A to 100% B at a rate of 2 ml/min in an SP column, and the fractions including the bFGF protein of about 18 KDa size were collected. Then, 500 mM NaCl solution A in 20 mM sodium phosphate (pH 7.0) buffer solution and 2M NaCl B in 20 mM sodium phosphate (pH 7.0) buffer solution were spilled to elute in a linear gradient from 0% A to 100% B at a rate of 2 ml/min in a heparin affinity column, and the fractions including the bFGF protein of about 18 KDa size were collected. At this time, fractions including bFGF were confirmed by SDS-PAGE analysis and then the quantification was performed.
(44) Molecular Modeling
(45) A candidate disulfide bondable group was set using 1BLA (NMR), which is a structure of proteins registered in the PDB. By using a protein contact map visualization program, the residues with C-alpha carbon distance of two amino acids of 7 Å or less and C-beta carbon distance of 5 Å were analyzed by using plot. Then, the formation of disulfide bonds were analyzed using a Yasara energy minimization server and performed energy minimization using AMBER force filed FF99 of chimera. Thereafter, the structure of the prepared protein was aligned with the wild-type bFGF to use the structure having the RMSD measurement value of 0.5 or less for experiments.
(46) CD (Circular Dichroism)
(47) For the structural analysis and TM measurement of wild-type bFGF and mutants, bFGF was dissolved in 20 mM sodium phosphate (pH 7.0), and the final concentration was adjusted constant to 0.2 mg/ml. In addition, it was put in a 0.1 cm cell, and the structure was analyzed under the conditions of the band width 1 nm in a 190 nm to 250 nm region, response 0.25 sec, data pitch 0.1 nm, scanning speed 20 nm/min, cell length 1 cm, accumulation 8 times, and temperature 20° C. In order to analyze the temperature stability, the melting temperature was performed at a 205 nm wavelength at 20° C. and 95° C. in 0.1 cm cell and 0.2 mg/ml concentration. Conditions were measured at 20° C. to 95° C. under the condition of 1° C./min.
(48) Residue numbers and predicted results for disulfide bond are exhibited in Table 1.
(49) TABLE-US-00001 TABLE 1 Disulfide Disulfide Disulfide prediction (alpha carbon) (beta carbon) RMSD 34-67 ◯ 4.6 3.9 0.397 34-70 ◯ 6.5 4.7 0.414 34-84 ◯ 6.7 4.6 0.353 40-82 ◯ 6.2 4.3 0.38 50-69 X 5.7 4.9 0.385 52-68 X 6 4.6 0.367 75-92 ◯ 4.9 3.9 0.395 76-108 ◯ 6.2 4.9 0.403 117-136 X 5.1 3.9 0.386 117-137 ◯ 4.5 4.5 0.403
(50) Cell Proliferation Assay
(51) In order to confirm whether the produced wild-type bFGF and the mutant actually exhibit activity, an experiment using cell proliferation ability was carried out by Genewel Inc. NIH-3T3 cells used for the experiment were maintained in a DMEM complete medium including 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. 2×10.sup.3 cells/well of NIH-3T3 cells were seeded in a 96 well culture plate. 24-hour cultured NIH-3T3 cells were starvated in a serum-free DMEM medium and then treated with the sample solution in DMEM medium including 0.5% FBS per concentration, and cultured for 72 hours. After culturing, 10 μl of MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] solution was added and reacted for 2 hours. Formazan crystal was dissolved with 100 μl of DMSO. Absorbance was measured at a wavelength of 540 nm using a spectrophotometer. The susceptibility to the drug was compared by the percentage of the absorbance of the drug untreated well (control) in the drug treated wells.
(52) Incubation Test
(53) 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 at 0.5 mg/ml in 1×PBS (pH 7.3) and incubated at 37° C., 50° C. and 60° C. water baths. They were sampled in the unit of 24 hours, and then centrifuged at 13000 rpm for 15 minutes at 4° C. to obtain only a supernatant. Through nano drop, the quantification and HPLC analysis were performed.
Example 1: Construction of pSSB-bFGF Plasmid Including Human bFGF cDNA
(54) DNA encoding bFGF was prepared by polymerase chain reaction using a human mononuclear cell cDNA library as a template and a primer. The base sequence of the primers used is as follows:
(55) TABLE-US-00002 Sense primer (SEQ ID NO: 3) 5′-GGCGGGCATATGCCCGCCTTGCCCGAGG-3′ and antisense primer (SEQ ID NO: 4) 3′-TGATGAGGATCCTCATCAGCTCTTAGCAGACAT-5′.
(56) The bFGF portion of
Example 2: Preparation of Escherichia coli Transformants of Human bFGF
(57) 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 medium (LB/ampicillin). After culturing for 12 hours at 37° C., it was mixed with 100% glycerol in a ratio of 1:1 and a stock was stored at −70° C.
Example 3: Purification of Human bFGF
(58) The stock prepared in Example 2 was inoculated into 10 ml of LB medium (LB/ampicillin) and cultured for 12 hours or longer. Then, the cells were transferred to 500 ml of LB medium (LB/ampicillin) and IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added so that a final concentration became 0.5 mM at an absorbance of O.D 0.4 to 0.5 at 600 nm. The cells were shaking cultured at 200 rpm for 4 hours at 37° C., and then centrifuged at 8000 rpm for 10 minutes to obtain E. coli pellets. The pellet was suspended in 25 ml of 20 mM sodium phosphate (pH 7.0) buffer solution, and the cells were disrupted by an ultrasonication method.
(59) The cell lysate disrupted by a ultrasonication method was centrifuged at 13000 rpm for 15 minutes at 4° C. The supernatant was collected and filtered using a 0.45 μm filter. The solution was purified by FPLC (Fast Performance Liquid chromatography), SP column and Heparin column. The conditions for purification were as follows: 100 mM NaCl solution A in 20 mM sodium phosphate (pH 7.0) buffer solution and 2 M NaCl B in 20 mM sodium phosphate (pH 7.0) buffer solution were spilled to elute in a linear gradient from 0% A to 50% B at a rate of 2 ml/min in an SP column, and the fractions including the bFGF protein of about 18 KDa size were collected. Then, 100 mM NaCl solution A in 20 mM sodium phosphate (pH 7.0) buffer solution and 2M NaCl B in 20 mM sodium phosphate (pH 7.0) buffer solution were spilled to elute in a linear gradient from 50% A to 100% B at a rate of 2 ml/min in an SP column, and the fractions including the bFGF protein of about 18 KDa size were collected. At this time, fractions including bFGF were confirmed by SDS-PAGE analysis and then the quantification was performed to obtain 10 mg of bFGF.
Example 4: Construction of pSSB-bFGF Mutant Plasmid
(60) pSSB-bFGF mutant plasmids were prepared by PCR using pfuUltraTMHF DNA polymerase as the template for the wild-type pSSB-bFGF plasmid and two complementary primers corresponding to the respective mutants. Then, the wild-type pSSB-bFGF plasmid, which was a template, was digested with Dpn I and transformed into E. coli Top10 by heat shock. Colonies resistant to ampicillin generated in the solid medium after transformation were selected and inoculated into 10 ml of LB medium (LB/ampicillin). After culturing for 16 hours at 37° C., DNA prep was performed and sequencing of the DNA obtained by DNA prep confirmed pSSB-bFGF mutant plasmids. The base sequence of the primers used is provided as follows:
(61) sense primer 5′-TCT ATC AAA GGA GTG TCT GCT AAC CGT TAC CTG-3′ (SEQ ID NO: 5) and the antisense primer 3′-CAG GTA ACG GTT AGC AGA CAC TCC TTT GAT AGA-5′ (SEQ ID NO: 6) at the substitution of the 69th cysteine codon TGT with serine codon TCT;
(62) sense primer 5′-TTA CTG GCT TCT AAA TCT GTT ACG GAT GAG TGT-3′ (SEQ ID NO: 7) and the antisense primer 3′-ACA CTC ATC CGT AAC AGA TTT AGA AGC CAG TAA-5′ (SEQ ID NO: 8) at the substitution of the 89th cysteine codon TGT with serine codon TCT;
(63) sense primer 5′-GCT AAC CGT TAC CTG TGC ATG AAG GAA GAT GGA-3′ (SEQ ID NO: 9) and the antisense primer 3′-TCC ATC TTC CTT CAT GCA CAG GTA ACG GTT AGC-5′ (SEQ ID NO: 10) at the substitution of the 75th alanine codon GCT with serine codon TCT;
(64) sense primer 5′-AAG CGG CTG TAC TGC TGC AAC GGG GGC TTC TTC-3′ (SEQ ID NO: 11) and the antisense primer 3′-GAA GAA GCC CCC GTT GCA GCA GTA CAG CCG CTT-5′ (SEQ ID NO: 12) at the substitution of the 26th lysine codon AAA with cysteine codon TGC;
(65) sense primer 5′-GGC TTC TTC CTG CGC TGC CAC CCC GAC GGC CGA-3′ (SEQ ID NO: 13) and the antisense primer 3′-TCG GCC GTC GGG GTG GCA GCG CAG GAA GAA GCC-5′ (SEQ ID NO: 14) at the substitution of the 34th isoleucine codon ATC with cysteine codon TGC;
(66) sense primer 5′-CAC CCC GAC GGC CGA TGC GAC GGG GTC CGG GAG-3′ (SEQ ID NO: 15) and the antisense primer 3′-CTC CCG GAC CCC GTC GCA TCG GCC GTC GGG GTG-5′ (SEQ ID NO: 16) at the substitution of the 40th valine codon GTT with cysteine codon TGC;
(67) sense primer 5′-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) at the substitution of the 50th histidine CAC with cysteine codon TGC;
(68) sense primer 5′-AGC GAC CCT CAC ATC TGC CTA CAA CTT CAA GCA-3′ (SEQ ID NO: 19) and the antisense primer 3′-TGC TTG AAG TTG TAG GCA GAT GTG AGG GTC GCT-5′ (SEQ ID NO: 20) at the substitution of the 52th lysine codon AAG with cysteine codon TGC;
(69) sense primer 5′-AAC CGT TAC CTG GCT TGC AAG GAA GAT GGA AGA-3′ (SEQ ID NO: 21) and the antisense primer 3′-TCT TCC ATC TTC CTT GCA AGC CAG GTA ACG GTT-5′ (SEQ ID NO: 22) at the substitution of the 76th methionine codon ATG with cysteine codon TGC;
(70) sense primer 5′-ACC AGT TGG TAT GTG TGC CTG AAG CGA ACT GGG-3′ (SEQ ID NO: 23) and the antisense primer 3′-CCC AGT TCG CTT CAG GCA CAC ATA CCA ACT GGT-5′ (SEQ ID NO: 24) at the substitution of the 117th alanine codon GCA with cysteine codon TGC;
(71) sense primer 5′-GTT GTG TCT ATC AAA TGC GTG TCT GCT AAC CGT-3′ (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) at the substitution of the 67th glycine codon GGA with cysteine codon TGC;
(72) sense primer 5′-GTG TCT ATC AAA GGA TGC TCT GCT AAC CGT TAC-3′ (SEQ ID NO: 27) and the antisense primer 3′-GTA ACG GTT AGC AGA GCA TCC TTT GAT AGA CAC-5′ (SEQ ID NO: 28) at the substitution of the 68th valine codon GTG with cysteine codon TGC;
(73) sense primer 5′-ATC AAA GGA GTG TCT TGC AAC CGT TAC CTG GCT-3′ (SEQ ID NO: 29) and the antisense primer 3′-AGC CAG GTA ACG GTT GCA AGA CAC TCC TTT GAT-5′ (SEQ ID NO: 30) at the substitution of the 70th alanine codon GCT with cysteine codon TGC;
(74) sense primer 5′-AAG GAA GAT GGA AGA TGC CTG GCT TCT AAA TCT-3′ (SEQ ID NO: 31) and the antisense primer 3′-AGA TTT AGA AGC CAG GCA TCT TCC ATC TTC CTT-5′ (SEQ ID NO: 32) at the substitution of the 82th leucine codon TTA with cysteine codon TGC;
(75) sense primer 5′-GAT GGA AGA TTA CTG TGC TCT AAA TCT GTT ACG-3′ (SEQ ID NO: 33) and the antisense primer 3′-CGT AAC AGA TTT AGA GCA CAG TAA TCT TCC ATC-5′ (SEQ ID NO: 34) at the substitution of the 84th alanine codon GCT with cysteine codon TGC;
(76) sense primer 5′-TAC AAT ACT TAC CGG TGC AGG AAA TAC ACC AGT-3′ (SEQ ID NO: 35) and the antisense primer 3′-ACT GGT GTA TTT CCT GCA CCG GTA AGT ATT GTA-5′ (SEQ ID NO: 36) at the substitution of the 108th serine codon TCA with cysteine codon TGC;
(77) sense primer 5′-GGA CCT GGG CAG AAA TGC ATA CTT TTT CTT CCA-3′ (SEQ ID NO: 37) and the antisense primer 3′-TGG AAG AAA AAG TAT GCA TTT CTG CCC AGG TCC-5′ (SEQ ID NO: 38) at the substitution of the 136th alanine codon GCT with cysteine codon TGC;
(78) sense primer 5′-CCT GGG CAG AAA GCT TGC CTT TTT CTT CCA ATG-3′ (SEQ ID NO: 39) and the antisense primer 3′-CAT TGG AAG AAA AAG GCA AGC TTT CTG CCC AGG-5′ (SEQ ID NO: 40) at the substitution of the 137th isoleucine codon ATA with cysteine codon TGC;
(79) sense primer 5′-GGG CAG AAA GCT ATA TGC TTT CTT CCA ATG TCT-3′ (SEQ ID NO: 41) and the antisense primer 3′-AGA CAT TGG AAG AAA GCA TAT AGC TTT CTG CCC-5′ (SEQ ID NO: 42) at the substitution of the 138th leucine codon CTT with cysteine codon TGC; and
(80) sense primer 5′-TTT CTT CCA ATG TCT TGC AAG AGC TGA TGA-3′ (SEQ ID NO: 43) and the antisense primer 3′-TCA TCA GCT CTT GCA AGA CAT TGG AAG AAA-5′ (SEQ ID NO: 44) at the substitution of the 144th alanine codon GCT with cysteine codon TGC.
(81) sense primer 5′-GAG AAG AGC GAC CCT TAT ATC AAG CTA CAA CTT-3′ (SEQ ID NO: 45) and the antisense primer 3′-AAG TTG TAG CTT GAT ATA AGG GTC GCT CTT CTC-5′ (SEQ ID NO: 46) at the substitution of the 50th histidine codon CAC with tyrosine codon TAT.
Example 5: Production and Purification of bFGF Mutants
(82) Each of the expression plasmids of bFGF mutants was transformed into E. coli BL21 (DE3) in the same manner as in Example 2, staked and cultured in 500 ml of LB medium (LB/ampicillin), and purified to obtain bFGF of about 18 KDa in size in the same manner as in Example 3. The amount of mutant thus obtained was variable according to the mutant, and about 4 to 12 mg of bFGF was obtained according to the mutant, and the purity was 98% or over.
(83) Each of the bFGF mutants is provided as follows:
(84) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 34th isoleucine and the 67th glycine are substituted with cysteine
(85) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 34th isoleucine and the 70th alanine are substituted with cysteine
(86) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 34th isoleucine and the 84th alanine are substituted with cysteine
(87) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 40th valine and 82nd leucine are substituted with cysteine
(88) A mutant 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
(89) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 50th histidine and the 69th cysteine are substituted with cysteine
(90) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 52nd lysine and the 68th valine are substituted with cysteine
(91) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 76th methionine and 108th serine are substituted with cysteine
(92) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 117th alanine and the 136th alanine are substituted with cysteine.
(93) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 117th alanine and the 137th isoleucine are substituted with cysteine.
(94) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 75th alanine is substituted with cysteine.
(95) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 26th lysine and 87th cysteine are substituted with cysteine.
(96) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 138th leucine is substituted with cysteine.
(97) A mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 are substituted with serine, and the 52nd lysine and 144th alanine are substituted with cysteine.
(98) A 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 50th histidine is substituted with tyrosine.
(99) The purification of the wild type and mutant can be purified through SP-sephrose and haparin affinity column. Both species were eluted at about 400 mM NaCl concentration in the SP column and 1.5M NaCl in the heparin column. After progressing the final heparin affinity column purification, SDS Page analysis was performed.
(100) As illustrated in
(101)
Experimental Example 1: Structural Analysis of Wild Type and Mutant bFGF Using Circular Dichroism
(102) The structure and thermal stability of the purified bFGF mutants of Example 5 were measured by circular dichroism analysis using a J-810 spectrometer (JASCO). The wild-type bFGF was purified using the purified bFGF in Example 3. For structural analysis, each bFGF is dissolved in 20 mM sodium phosphate (pH 7.0), and the final concentration is adjusted constantly to 0.1 mg/ml. Then, the structure was analyzed under the conditions that it was put in a 0.1 cm cell, and in 190 nm to 250 nm region, 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, and the temperature was 20° C.
(103) In order to analyze thermal stability, Tm (melting temperature) was compared with far-UV at 20° C. and 95° C. to determine the wavelength of 208 nm and 0.1 mg/ml concentration in 0.1 cm cell. Conditions were measured at 20° C. to 95° C. under the condition of 1° C./min. The results are exhibited in Table 2.
(104) TABLE-US-00003 TABLE 2 Mutant bFGF Structure Mutant bFGF Structure change Mutant bFGF Structure change Name change (Tm) Name (Tm) Name (Tm) Wild-type bFGF −(57.5° C.) Mutant A34- Reduced 48 Mutant B34-70( No change (SEQ ID NO.: 1) 67 Mutant C34-84 No change Mutant D40- No change Mutant E40-84 No change 82 Mutant F50-69 No change Mutant G52- No change Mutant H76-108 No change (SEQ ID NO.: 8) 68 (SEQ ID NO.: 9) Mutant I117-136 No change Mutant J117- No change Mutant K75 Change (62° C.) 137 Mutant L26-87 No change Mutant M138 No change Mutant N52-144 No change Mutant (65° C.) K75 + H50Y
(105) Table 2 exhibits the results of measurement of the degree of structural change for wild-type bFGF and bFGF mutants and the fraction unfolded per temperature at a wavelength of 208 nm in a circular dichroism analysis for measuring thermal stability. When the folding-loosening phenomenon occurs, the structure changes in the region around 208 nm is exhibited. Using this, the accurate Tm value was analyzed by measuring the melting temperature TM within the range of 20 to 95° C.
(106) 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 the respective corresponding locations are substituted with cysteines to induce intramolecular disulfide bonds.
(107) As a result, most of the structural changes exhibited the same structure as that of wild-type bFGF, and there was no change, and the mutants added with disulfide bonds had no specific structure. The Tm, which exhibits a thermal stability, is identical to that of the most bFGF mutants as compared to the wild-type bFGF at 58° C. Among them, 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 substituting one amino acid with cysteine.
(108) Meanwhile, in order to confirm the conspicuousness of the K75 mutant having the 69th and 87th cysteines of SEQ ID NO: 1, which are specific positions of the present disclosure, substituted with serine and the 75th alanine further substituted with cysteine, the thermal stability of the wild-type bFGF of SEQ ID NO: 1, the bFGF mutant (Cys.fwdarw.Ser mutant) in which only the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine, the a bFGF mutant (wild type+disulfide bond) in which only the 75th alanine of SEQ ID NO: 1 was substituted with cysteine and the K75 mutant (Cys.fwdarw.Ser mutant+disulfide bond, the bFGF mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine, and the 75th alanine was substituted with cysteine) was compared and confirmed.
(109) As a result, as illustrated in
(110) In a further experiment, it was confirmed that the TM of thermal stability of HsbFGF in which the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine, and the 50th histidine was substituted with tyrosine in the K75 mutant in which the 75th alanine was further substituted with cysteine was improved up to 65° C. compared to the wild-type bFGF at 58° C. and K75 mutant at 62° C. This means that one amino acid existing on the surface is substituted with tyrosine 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.
(111) As a result, as illustrated in
Experimental Example 2: Examination of Cell Proliferation of Wild-Type and Mutant bFGF
(112) The made wild-type bFGF and bFGF mutants having the structure of using solubility and circular dichroism and showing good results through the result analysis of TM were selected to perform a cell proliferation examination. Cell proliferation examinations were entrusted to Genewel Inc., and were performed with NIH3T3 cell line, a skin cell sensitive to bFGF. As an experiment method, NIH-3T3 cells were maintained in DMEM complete medium including 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. NIH-3T3 cells of 2×10.sup.3 cells/well were seeded in a 96 well culture plate. 24-hour-cultured NIH-3T3 cells were treated with serum-free DMEM medium and treated with sample solution in DMEM medium including 0.5% FBS per each concentration after starvation, and then was cultured for 72 hours. After culturing, 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 used to dissolve formazan crystal. Absorbance was measured at a wavelength of 540 nm using a spectrophotometer. The susceptibility to the drug was compared by the percentage of the absorbance of the untreated well (control) in the drug treated wells. As illustrated in
Experimental Example 3: Quantitative Analysis of Protein by Incubation of Wild-Type and Mutant bFGF
(113) In order to confirm the stability of the wild-type bFGF and mutants [bFGF mutant (Cys.fwdarw.Ser mutant) in which only the 69th and 87th cysteine of SEQ ID NO: 1 were substituted with serine, bFGF mutant in which only the 75th alanine of SEQ ID NO: 1 was substituted with cysteine (wild type+disulfide bond) and the K75 mutant (Cys.fwdarw.Ser mutant+disulfide bond, bFGF mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine and the 75th alanine was substituted with cysteine), 37° C. short-term incubation test was performed. In the state of PBS (phosphate buffer saline), which is the most similar to the human body, the wild-type bFGF and its mutants were dissolved at 0.5 mg/ml and incubated in a water bath at 37° C. They were sampled in the unit of 48 hours, 7 days, and 10 days and centrifuged at 13000 rpm for 15 minutes at 4° C. to obtain only supernatant. Proteins were quantified using Nano drop. As time goes by, the concentrations of wild-type bFGF and mutants were quantitatively determined, and the wild-type bFGF exhibited a more significant decrease than the mutant. The results are exhibited in Table 3.
(114) TABLE-US-00004 TABLE 3 Day After After one After bFGF name 0 48 h week 10 days Wild type 100 56 26 16 CYS .fwdarw. SER mutant 100 88 80 62 Wild type + disulfide 100 92 78 64 K75 (CYS .fwdarw. SER 100 90 88 80 mutant + disulfide bond)
(115) In addition, based on the above results, the determination of whether the K75 mutant had long-term storage stability in comparison with the wild-type bFGF in the PBS (phosphate buffer saline) state, which is the most similar to the human body using the K75 mutant having increased thermal stability was confirmed. First, the wild-type bFGF and K75 mutants were dissolved in PBS (phosphate buffer saline) at the same concentration, followed by incubation at 25° C. for 20 days. After centrifugation, the supernatant was quantitatively analyzed using HPLC.
(116) As a result, as illustrated in
Experimental Example 4: HPLC Analysis of Wild-Type and Mutant bFGFs at 50 and 60° C. Cincubation
(117) In order to confirm the stability of the wild-type bFGF and K75 mutants (Cys.fwdarw.Ser mutant+disulfide bond, bFGF mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine and the 75th alanine was substituted with cysteine) and the HsbFGF K75 mutant (Cys.fwdarw.Ser mutant+disulfide bond, bFGF mutant in which the 69th and 87th cysteines of SEQ ID NO: 1 were substituted with serine, the 75th alanine was substituted with cysteine, and the 50th histidine was substituted with tyrosine), the incubation test was performed for a week at 50° C. and for 5 days at 60° C. Each of the wild-type 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 according to the dates were centrifuged at 13000 rpm for 15 min at 4° C. to obtain only supernatant, and the protein was analyzed by using HPLC and UV spectrometer.
(118) As a result, as illustrated in
(119) Also, as illustrated in
(120) In the HPLC analysis using the results illustrated in