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

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

DOI：10.3969/j.issn.1673-1689.2012.12.006

中文关键词: 干扰素α2b(IFNα2b) 人血清白蛋白(HSA) 毕赤酵母 质量检测 融合蛋白

英文关键词: interferon-alpha 2b(IFN α2b) human serum albumin(HSA) Pichia pastoris quality detection fusion protein

基金项目:国家自然科学基金项目(30970029)

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中文摘要:

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5 L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85 821,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1.6×106IU/mg。具有良好的工业应用价值。

英文摘要:

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.

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