重组瑞替普酶包涵体制备及其体外复性  

Preparation of recombinant reteplase inclusion bodies in E. coli and its refolding in vitro

作　　者：王俊雄[1] 关怡新[1] 姚善泾[1] 

机构地区：[1]浙江大学化学工程与生物工程学院,浙江杭州310027

出　　处：《化工学报》2015年第2期709-716,共8页CIESC Journal

基　　金：国家自然科学基金项目(21036005)~~

摘　　要：将携带重组瑞替普酶(reteplase,rt-PA)的质粒成功转化到大肠杆菌BL21(DE3)后,诱导表达获得包涵体,考察了诱导剂浓度、培养温度和培养时间等条件对目标蛋白表达量的影响。在此基础上,对高效表达的rt-PA包涵体体外复性过程进行了详细研究。首先利用单因素实验考察了复性液p H、GSH浓度、GSH/GSSG比例、蛋白浓度等各种复性条件对复性效果的影响;并结合正交实验设计,进一步研究了高蛋白浓度下复性后rt-PA酶活变化情况。以0.2 mmol·L-1 IPTG诱导,在33℃下培养6 h,每升发酵液约可获得1.7 g粗制包涵体。适宜的复性条件为蛋白浓度50μg·ml-1,p H 10.0,GSH浓度1 mmol·L-1,GSH/GSSG比例8,复性收率为87.2%。影响高蛋白浓度下rt-PA复性的关键因素为复性液初始p H及GSH浓度,在800μg·ml-1蛋白浓度下复性后rt-PA比活可达7.54×104 IU·mg-1,荧光光谱分析结果表明复性后rt-PA恢复了其天然态结构。The vector with the gene of recombinant reteplase（rt-PA） was cloned into E. coli BL21（DE3） cells. Over-expression of rt-PA as inclusion bodies was obtained, and renaturation of rt-PA in vitro was investigated. Firstly, single factor experiments were conducted to optimize refolding conditions, including refolding buffer p H, GSH concentration, ratio of GSH to GSSG, rt-PA concentration. On this basis, high concentration protein refolding was further investigated by orthogonal experimental design. Fermentation broth with 1.7 g crude inclusion bodies per liter was obtained after using 0.2 mmol·L^-1 IPTG as inducer and culturing at 33℃ for 6 h. The refolding yield of rt-PA was up to 87.2% under optimal condition： 50 μg·ml-1 denatured rt-PA, p H 10.0, 1 mmol·L^-1 GSH and ratio of GSH to GSSG 8. The key factors affecting refolding of high concentration protein were initial p H and GSH concentration, and specific bioactivity of rt-PA could reach 7.54×104 IU·mg^-1 after refolding at protein concentration of 800 μg·ml^-1. Fluorescence spectra indicated that structural conformation of refolded reteplase was identical with its native state.

关 键 词：生物分离 蛋白质复性 二硫键 正交实验设计 瑞替普酶 

分 类 号：TQ028.8[化学工程]