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Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.
Pichia pastoris expression system is similar to that of the mammalian cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylate d haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.