【摘要】 对重组牛凝血酶原-2包涵体的体外重折叠复性进行了研究。凝血酶原-2是α-凝血酶生成过程中的一个最小的单链中间前体。重组牛凝血酶原-2在E.coli中高效表达并形成包涵体,经2.5mol?L?1脲和0.7%Triton X-100洗涤,再用8mol?L?1脲和0.3mol?L?1DTT溶解后,得到了包涵体裂解液。研究优化了体外复性溶液,其组成为含2.7mol?L?1脲、0.6mmol?L?1GSSG、GSSG/GSH0.9、0.1%PEG6000和0.5mol?L?1L-Arg、pH7.4Na3PO4缓冲液。牛凝血酶原-2复性后经透析,再被Echis Carinatus Snake Venom激活,转化为有活性的α-凝血酶,其总活力可达2948U?mL?1。结果表明稀释复性法可用于重组牛凝血酶原-2包涵体的体外重折叠复性。更多还原

【Abstract】 Prethrombin-2 is the smallest single-chain precursor to thrombin and has the same molecular weight with thrombin. The recombinant bovine prethrombin-2 was expressed and overproduced in E.coli BL21(DE3) as intracellular insoluble inclusion bodies. After washed by 2.5mol?L?1 urea and 0.7% Triton X-100, solubilized by 8 mol?L?1 urea and 0.3 mol?L?1 DTT, the key parameters in the refolding process, such as GSSG concentration, GSSG/GSH, urea concentration, L-Arg concentration and operation temperature were investigated by linear regression orthogonal experiments combined with steepest ascent experiments. Good results are obtained when refolding buffer consisted of 2.7 mol·L-1 urea, 0.6m mol?L?1 GSSG, GSSG/GSH 0.9, 0.1%PEG6000, and 0.5 mol?L?1 L-Arg. After dialysed, the refolded prethrombin-2 was activated by Echis Carinatus snake venom and assayed by using peptide chromogenic and fluorogenic substrate S-2160 (Bz-L-Phe-L-Val-L-Arg-pNA). Using the refolding process with the above mentioned parameters, the total activity of the activated refolded prethrombin-2 is about 2948 U?mL?1 at optimum refolding process. The results indicate that the refolding process used is feasible to recover native activity of recombinant bovine prethrombin-2.更多还原