人血清白蛋白和干扰素α2b融合蛋白在毕赤酵母中表达及质量控制

筛选获得毕赤酵母工程菌GS115/pPIC9K/hsa-ifnα2b,胞外分泌表达rHSA-IFNα2b药物蛋白.经摇瓶及5L发酵罐条件优化,确定最佳发酵条件.经50 L发酵罐工艺放大,发酵产量稳定,可达到350 mg/L.离心获得发酵上清液,超滤浓缩10倍,再依次通过Blue Sepharose 6FF亲和层析、Phenyl Sepharose 6FF疏水层析及Q Sepharose 6FF离子柱纯化.纯化产品进行SDS-PAGE纯度检测、SEC-HPLC检测、相对分子质量测定、N端测序、内毒素检测和生物活性检测.筛选获得一株高产菌,优化获得最佳的发酵条件.纯化产品检测(SEC-HPLC)纯度大于95％,相对分子质量为85821,氨基酸的N端测序为DAHKSEVAHRFKDLG,与预期的相符.内毒素含量小于5EU/mg,符合国家药典的要求.体外生物细胞活性高达1.6×106 IU/mg.具有良好的工业应用价值.

Expression and Quality Control of the Recombinant Human Serum Albumin and Interferon-Alpha2b Fusion Protein in Pichia pastoris

The engineering pichia pastorios GS115 is constructed which express recombinant human serum albumin and interferon-alpha2b fusion protein.The fermentation conditions are optimized in flask and 5 L fermentor.The surpernatant was purified by Blue Sepharose 6FF affinity chromatography,Phenyl Sepharose 6FF hydrophobic chromatography and Q Sepharose 6FF ion-exchange chromatography.The purified protein was detected by SDS-PAGE,molecular weight determination,nitrogen terminal sequence,endotoxin determination and anti-virus biological activity detection.The fermentation technology condition is stable and the harvest can reach 350 mg/L.The purity of purification protein is more than 95%.Molecular weight is 85 820.9 Da and amino acid sequence of nitrogen terminal is DAHKSEVAHRFKDLG.The result is correspond to the expected.The endotoxin is less than 5 EU/mg,which meet the standard requirement.The anti-virus activity of the protein is 1.6×106 IU/mg by bio-assay.It has excellent properties for industrial applications.