A Recombinant Human Acidic Fibroblast Growth Factor Expression, Purification And Characterization In E.coli, S.cerevisiae And P.pastoris

Acidic fibroblast growth factor (aFGF), a member of a family of structurally related polypeptides, is a kind of multifunctional protein that can stimulate cellular proliferation, migration and differentiation. In vivo, aFGF has angiogenic activity promoting vascular endothelial cell mitogenesis as well as chemotaxis and induction of proteases involved in tissue regeneration. Due to this wide range of biological activities, aFGF was thought to be an potential therapeutic drug in acceleration of wound healing, prevention of restenosis after angioplasty, regeneration of nerves, cartilage, and bone tissue, and the healing of gastric ulcers as well as retinal and tympanic membranes. Since the source of natural human aFGF (haFGF) is too limited to meet clinical demanding, attempts have been made to produce haFGF in a large scale by recombinant DNA technique. The haFGF gene cloned and expressed in E.coli has been reported, but the research on its expressed in eukaryotic cell is few. To obtain a haFGF with better properties than that expressed by E.coli, mutation was applied to changing the construction of haFGF. But this attempt did not succeed in improving the function of haFGF. Sequence analysis deduced that there were some potential sites for glycosylation within haFGF gene. Glycosylation has been proved to affect properties of proteins, which might endue some new properties to haFGF. In this study we constructed two yeast expression systems: S.cerevisiae and P.pastoris which had the ability to glycosylation of heterogenous proteins. The reasons for choosing yeast expression system are that yeast is an ideal host for expression of exogenous gene with the advantages of both prokaryocyte and eukaryocyte, the haFGF expression by yeast cells produce glycosylation or unglycosylation modification that may change some properties of haFGF and improve its' function.Methods: First of all, haFGF gene was isolated from woman endometrium by RT-PCR and inserted into clone vector. Then haFGF gene was isolated with restriction enzyme from clone vector and subcloned into the expression vectors. The new construct pET-30a(+)-haFGF, was transformed into the E.coli strain BL21(DE3) by CaCl2 method. Recombinant plasmid pYES2-haFGF was transformed into S. cerevisiae strain INVSc1 by PEG method. pPIC9K-haFGF was linearized by SalI and then transformed into the P. pastoris strain KM71 by electroporation method. After screening high expression level recombinants, recombined haFGFs were induced and isolated. Condition of inductionwas optimized and inspected by ELISA. Recombined proteins were purified by heparin-Sepharose affinity chromatography and gel filtration chromatography. The purity of recombined protein was clarified by SDS-PAGE and western blot. The biological activity of the rhaFGF expressed by P. pastoris was investigated by MTT assay using NIH3T3. Some other characters of rhaFGFs, which expressed by P. pastoris, were also investgated such as the stability and ability of promoting smooth muscle cell (SMC) and endothelial cell proliferation. Experiment Results: 1.Two haFGF genes were cloned from endometrium by RT-PCR. One is composed of three extrons, which named full-length haFGF. The other is composed of two extrons. 2. Using pET-30a(+) as expression vector, BL21(DE3) as host strain , successfully constucted E.coli /haFGF. The produce of rhaFGF(E) (Recombined human acidic fibroblast growth factor expressed by E.coli) was induced by IPTG. In shake-flask fermentation, the production of rhaFGF(E) peaks within 3 hours. Targeting protein was obtained from supernatant of cell lysate by heparin-Sepharose affinity chromatography. SDS-PAGE and western blot analysis showed the purification procedure as mentioined above yielded highly purified protein. After being digested out 6×His tag , the protein was observed as one clear band in the SDS-PAGE gel with the same molecular weight as natural haFGF. The immunoreactivity was also confirmed by Western blotting.3. Succeed in recombinantation of S.cerevisiae /haFGF usi...