High-level expression, purification, and characterization of recombinant human basic fibroblast growth factor in Pichia pastoris.
Basic fibroblast growth factor [basic FGF (bFGF); FGF-2] is an important member of the FGF family, bFGF is a potent angiogenic molecule in vivo and in vitro stimulate smooth muscle cell growth, wound healing, and tissue repair. The full-length hbFGF coding sequence, gained by RT-PCR, was cloned into the pPICZalphaA vector in frame with the yeast alpha-factor secretion signal under the transcriptional control of the AOX promoter and integrated into Pichia pastoris strain X33, and the high level expression of rhbFGF has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that rhbFGF, an 18 kDa protein, was secreted into the culture medium. The growth conditions of the transformant strain were optimized in 50 ml conical tubes including methanol concentration, pH and inducing time. Under the optimal conditions, stable production of rhbFGF around 150 mg/l was achieved. The expressed rhbFGF was purified more than 94% purity using SP Sepharose ion exchange chromatography and source 30RPC. A preliminary biochemical characterization of purified rhbFGF was performed by biological activity analysis which was used by NIH/3T3 cell cultures, and the results demonstrated that the purified rhbFGF stimulated the growth of NIH/3T3 cells similarly to standard material.
Mu
Xupeng
X
Department of Biochemistry, Institute of Frontier Medical Sciences, Jilin University, Xinmindajie Street 1163, Changchun, Jilin 130021, China.
Kong
Ning
N
Chen
Weili
W
Zhang
Ting
T
Shen
Mohan
M
Yan
Weiqun
W
D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D041681:NIH 3T3 Cells; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology
D003470:Culture Media; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2