共表达TrxA和DsbC对富含二硫键的异源蛋白在大肠杆菌中表达的影响

以复制子为p15A的质粒pACU184为基础 ,构建了 3种表达硫氧还蛋白 (TrxA)或 和二硫键异构酶 (DsbC)的表达质粒 .经IPTG诱导 ,克隆的DsbC和TrxA都以可溶的形式高表达 .分别将构建的 3种表达质粒与复制子为colE1并克隆有人源化鼠抗人纤维蛋白单链抗体 低分子量尿激酶融合基因 (C6 UK)的表达质粒共转化大肠杆菌XL1 blue ,在 30℃用IPTG诱导表达 .SDS PAGE显示 ,共表达TrxA或DsbC都能导致C6 UK融合蛋白的部分可溶性表达 ,而且同时共表达TrxA和DsbC 2种分子时 ,C6 UK完全以可溶形式表达 ,但表达量降低 .分别用溶圈法和ELISA检测了各种共表达时可溶表达产物的生物活性 .结果显示 ,只有共表达DsbC时才能检测到明显的C6 UK融合蛋白的双功能

The Influence of Coexpression of TrxA and DsbC to the Expreesion of Heterogenous Protein with Multiple Disulfide Bonds

Based on the plasmid pACU184 that carried the origin of replication from plasmid p15A, three expression vectors that could express disulfide isomerase(DsbC) and/or thioredoxin(TrxA) were constructed. After induced with IPTG, cloned DsbC and TrxA could be expressed in soluble form with high level in E.coli. These three vectors were respectively transformed into E.coli XL1 blue together with a compatible expression vector for the fused gene of humanized mouse anti human fibrin ScFv and low molecular weight single chain urokinase (C6 UK). With the inducement of 1 mmol/L IPTG at 30℃,SDS PAGE showed that C6 UK was expressed in partially soluble form with co expression of either DsbC or TrxA, but it was expressed in totally soluble form with co expression of both DsbC and TrxA. The expression level in the latter case was lower than the former. By the methods of dissolved circle and ELISA, the bioactivity of C6 UK was assayed for different co expressions. The results clearly showed that bioactive fused proteins C6 UK were only obtained by co expression of DsbC.