Abstract:

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 pH, 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 mg·ml^-1 denatured rt-PA, pH 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 pH and GSH concentration, and specific bioactivity of rt-PA could reach 7.54×10⁴ IU·mg^-1 after refolding at protein concentration of 800 mg·ml^-1. Fluorescence spectra indicated that structural conformation of refolded reteplase was identical with its native state.

Key words: bio-separation, protein refolding, disulfide bond, orthogonal experimental design, reteplase

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pH、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50 mg·ml^-1,pH 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pH及GSH浓度,在800 mg·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。

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