共表达折叠调节因子对尿激酶功能性表达的影响

Effects of co-expression of protein folding modulators on activity of urokinase expressed in E. coli

目的研究共表达折叠调节因子对真核蛋白尿激酶(urokinase,UK)功能性表达的影响,改善大肠杆菌中UK表达产物的活性。方法通过分离分子伴侣DnaK、DnaJ、GroEL-S及二硫键异构酶DsbA、DsbC的基因,构建折叠调节因子表达载体,与UK表达载体双质粒共转化同一个菌株,观察共表达与单独表达时UK的表达产物活性变化。结果共表达分子伴侣DnaK、GroEL-S和二硫键异构酶DsbC、DsbA对UK表达产物的活性有明显的提高,最高的是GroEL-S。结论在大肠杆菌中共表达分子伴侣能协助尿激酶蛋白的正确折叠,提高尿激酶表达产物的活性。

Objective To study the coexpression of protein folding modulators, including chaperones （DnaK, DnaJ, and GroEL-GroES） and disulfide isomerase Dsb molecules （DsbA and DsbC） on the bioactivity of eukaryotic expressed urokinase （UK）. Methods The gene sequences of DnaK, DnaJ, GroEL-GroES, DsbA and DsbC were isolated from the chromosome of E. coli by PCR. The obtained fragments mentioned above were cloned into the plasmid pBAD-1 under the control of arab promoter and then the plasmids were transformed into E. coli BW25113 and HB101 together with a compatible vector pQE-UK. The amount of expressed UK was measured by Bradford method and its activity was analyzed by fibrin plate method for its fibrinolysis. Results The activity of expressed UK was increased by co-expressing with DnaK, GroEL-GroES, DsbA, and DsbC in BW25113. Among them, the highest activity was achieved by co-expressing with GroEL-GroES. Conclusion Co-expressing of chaperones or disulfide isomerase can improve the bioactivity of UK expressed in E. coll.