组织因子途径抑制因子重组蛋白表达和纯化方法的优化  

Optimization of Expression Conditions for Purification of Recombinant Tissue Factor Pathway Inhibitors

作　　者：韩彦峰[1] 刘兰霞[1] 宋丽萍[1] 董霞[1] 张海玲[1] 冷希岗[1] 

机构地区：[1]中国医学科学院、北京协和医学院生物医学工程所,300192

出　　处：《天津医药》2009年第1期13-16,共4页Tianjin Medical Journal

基　　金：天津市科技攻关培育项目(项目编号:06YFGPSH03700)

摘　　要：目的:对组织因子途径抑制因子(TFPI)的表达条件和纯化方法进行优化,降低重组蛋白制备成本。方法:应用巴斯德毕赤酵母表达系统进行TFPI重组蛋白的表达,采用不同浓度甲醇进行重组蛋白表达诱导,观察不同pH对重组蛋白活性的影响,应用硫酸铵分级沉淀、阴离子交换层析和凝胶过滤层析相结合的方法纯化重组蛋白。结果:重组蛋白的表达量达到1.5mg/L培养基。上述蛋白纯化方法所得回收率为43.1%,蛋白纯度大于95%,蛋白活性保持良好。结论:优化后的蛋白表达量比优化前得到明显提高,本研究采用的纯化方法操作简单、耗时短、成本低且回收率较高。Objective： To optimize the expression condition for purification of recombinant tissue factor pathway inhibitor （rTFPI）. Methods： TFPI was expressed in Pichia pastoris and the effects of the concentration of methanol and the pH value of the medium on the expression of rTFPI were observed. The rTFPI was purified by the combination of sodium sulfate precipitation, ion-exchange chromatography, and gel filtration. Rosults： The expression of rTFPI reached 1.5 mg per liter of culture medium, which was significantly higher than that of the expression procedure previously applied in our lab. The recovery rate of the eurrent purification procedure was 43.1% and the purity of the recombinant protein was around 95%. The purified rTFPI remained bioactive. Conclusion：The results showed that the expression of rTFPI was significantly elevated after the optimization of the induction condition. The current procedure for the purifieation of rTFPI was simple, more rapid, and cost effective.

关 键 词：凝血致活酶 重组蛋白质类 毕赤酵母 甲醇 氢离子浓度 

分 类 号：R318.08[医药卫生—生物医学工程] R318[医药卫生—基础医学]