人TNF-α突变体Y87H/A145R的表达及纯化  

Expression and Purification of Human TNF-α Variant Y87H/A145R

作　　者：李壮林[1] 姚雪静 于学珍 

机构地区：[1]山东大学微生物国家重点实验室,山东济南250100 [2]烟台荣昌生物工程有限公司,山东烟台264006

出　　处：《中国医药工业杂志》2011年第5期332-336,共5页Chinese Journal of Pharmaceuticals

摘　　要：用大肠杆菌表达人肿瘤坏死因子-α(TNF-α)的突变体Y87H/A145R,并研究其生物学特性。在5 L发酵罐中培养大肠杆菌工程菌,当菌体密度(D600)增长到35时,用异丙基硫代半乳糖苷(IPTG)诱导目标蛋白表达,包涵体蛋白的最终表达水平为2.5 g/L。用High Q Micro-prep凝胶柱纯化包涵体蛋白,然后通过透析复性,蛋白纯度大于99%。用分子排阻色谱分析Y87H/A145R的分子特征。结果表明,Y87H/A145R与TNF-α一样,以三聚体形式存在。而且,Y87H/A145R能明显抑制TNF-α诱导的L929细胞凋亡。小鼠试验表明,突变体Y87H/A145R不但失去了TNF-α的细胞毒性,并且能有效抑制TNF-α引起的急性肝坏死。Human tumor necrosis factor-α(TNF-α) variant Y87H/A145R was expressed in Escherichia coli for research on its biological characteristics.The engineered strains harboring Y87H/A145R gene were cultured in a 5 L fermenter.Y87H/A145R protein was over-expressed as inclusion body in E.coli after induction with IPTG.The expression level was estimated to be 2.5 g/L.Inclusion body protein was purified by High Q Micro-prep gel and refolded by dialysis.The molecular structure of Y87H/A145R was analyzed by molecular exclusion chromatography and the results showed that the variant could form a trimer as same as natural TNF-α.In addition,it was proved that Y87H/A145R could specifically inhibit apoptosis of L929 cells stimulated by TNF-α.Subsequent studies demonstrated that Y87H/A145R lost toxicity shown by TNF-α,and it significantly inhibited acute hepatic necrosis of mice stimulated by TNF-α.

关 键 词：人肿瘤坏死因子-Α 突变体 表达 纯化 包涵体 

分 类 号：R9[医药卫生—药学]