采用高密度发酵法在5.5 L发酵罐中对pPIC9-pEGF/GS115重组毕赤酵母Pichia pastoris菌株进行发酵,在培养至酵母菌体密度(D600nm)约150时,以甲醇诱导目的蛋白表达,48 h后菌体密度达315,目的蛋白表达产量约300 mg/L.发酵上清液经0.45μm和截留相对分子质量30 000的超滤膜、亲和层析、透析后目的产物的纯度达90%以上.鉴定结果表明:纯化的表达产物pEGF对BALB/c 3T3细胞具有显著的增殖作用.%The recombinant Pichia pastoris strain infected with pPIC9-pEGF/GS115 for expression of strain porcine epidermal growth factor was high cell density cultured in a 5.5 L fermentor. Following prefermentation and reaching D600nm density to 150, the yeast cells were induced by addition of methanol,which elevated cell density D600nm to 315. As the result, the medium concentration of secreted pEGF reached 300 mg/L. The pEGF fermentation product was purified to 90％ by ultrafiltration, His-tag affinity column chromatography and dialysis. In vitro biological activity test results showed that the expressed pEGF fusion protein could enhance significantly the proliferation of BALB/c 3T3 cells. The above results demonstrated successful preparation of biologically active pEGF by the P. pastoris eukaryotic expression system.
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