rPA(K)的原核表达及其产物的初步纯化

rPA(K)是经过缺失/定点突变技术所获得的t-PA突变体。本实验以IPTG诱导,实现了rPA(K)在原核系统中的表达。实验证明目的产物的表达形式为包涵体,且当以IPTG诱导3h时,表达量最高,为41%。此后对该表达产物进行了初步纯化,即通过对不同超声破碎次数,包涵体洗涤液(脲,Triton X-100,乙醇)的不同浓度对纯化的影响因素进行了摸索。结果表明,超声2次,2M脲,0.5%Triton X-100,20%乙醇对目的蛋白纯化的效果最好,从而为进一步的纯化和复性奠定基础。

Expression of rPA(K)in Prokaryotic System and The Preliminary Purification of Its Product

rPA(K)was the deleted mutant of t-PA.The experiment,through the induction of IPTG,made the expression of rPA(K)in prokaryotic system come true.Then it was certificated that the expression way of product was inclusion body,and the level of expression was the highest after the induction of IPTG for 3 hours.It reached 41%.Through the different times of ultransonication and different concentration of detergents(urea,Triton X-100,ethanol),the aim of this experiment was to grope the different kinds of factors and realize the preliminary purification of rPA(K).Thus the expressive protein which had formed inclusion body has been recovered its activity.The result indicated that two times of ultransonication,2urea,0.05% Triton X-100,20% ethanol was the best.By doing that,we hoped to get an effective and feasible way for the later purification and renaturation.