【摘要】 目的建立和优化重组人淋巴毒素α(rhLT-α)工程菌高密度发酵方法以及目的蛋白的复性和纯化方法,得到高纯度的重组人淋巴毒素α。方法高密度发酵rhLT-α工程菌,超声破碎细胞提取包涵体,经过复性后,用DEAE Sepharose FF,Phenyl Sepharose FF(LS)进行分离纯化。结果重组人淋巴毒素α以包涵体形式表达,经过破碎离心收集包涵体,洗涤后包涵体纯度达到85%以上。复性后,经两步纯化,最终目的蛋白纯度达98%以上。活性达6×108U/mg以上。结论建立了高效简便的淋巴毒素生产工艺,为淋巴毒素的规模生产打下了基础。更多还原

【Abstract】 Objective To establish the high-cell-density fermenting method of recombinant human lymphotoxin alpha in Escherichia Coli and the annealing and purification methods of objective protein,then optimize the methods and obtain high purity rhLT-α.Methods\ the engineering E.Coli was fermented in high-cell-density and induced by IPTG.Recombinant protein was refolded in vitro and purified by DEAE Sepharose FF anion chromatography and Phenyl Sepharose FF(LS) hydrophobic chromatography.Result\ The recombinant protein was expressed inclusion body,then the engineering E.Coli was split by ultrasonic,Inclusion body was harvested and washed,the purity of objective protein more than 85%.After annealing and two-step purifying,the purity of the final objective protein is more than 98% and the bioactivity is more than 6×10~8U/mg.Conclusion\ We established the high performance process of rhLT-α and this study provides a basis for the rhLTα industrial production.更多还原