星海链霉菌卤化酶重组蛋白在大肠杆菌中可溶表达

摘要

大肠杆菌(Escherichia coli ,E.coli)是表达异源蛋白的良好宿主,但常遇到蛋白表达产物不可溶的困难.在对来自星海链霉菌的卤化酶基因sinH 进行大肠杆菌异源表达时,为克服蛋白表达产物可溶性低的问题,探讨了不同载体对蛋白可溶表达的影响.利用 pET28a进行 SinH 表达时不能得到可溶的目标蛋白；使用含有泛素标签及内含肽的载体 pHUIE 实现了卤化酶蛋白 SinH 的可溶表达,但纯化后纯度不高；利用带有链霉菌分子伴侣蛋白基因的载体 pET28a-SinH 与 pETcoco-pL1SL2在 E.coli BL21(DE3)中共表达,在30℃,0.1 mmol/L IPTG诱导条件下表达4 h,成功获得大量可溶蛋白,使用亲和层析(Ni-NTA)纯化,获得了较纯的目标蛋白 SinH,上述研究结果为在大肠杆菌中进行其他链霉菌蛋白的可溶表达提供了参考.%Escherichia coli (E.coli)is a good host for expression of heterologous proteins,but insoluble protein products are often obtained.In order to overcome the problem of low soluble expression,halogenase gene sinH from Streptomyces xinghaiensis is expressed by E.coli,the effect of different vectors on the solubility is discussed.No soluble target protein is detected when it is expressed using pET28a,whereas by using expression vector pHUIE containing ubiquitin tag and intein,soluble target protein is obtained and purified,but the purity is low.The coexpression system containing pET28a-SinH and pETcoco-pL1SL2, which carries genes encoding Streptomyces chaperonin,is constructed in E.coli BL21(DE3),and the optimized induction condition is established as 0.1 mmol/L IPTG for induction,and expression time is 4 h under 30 ℃.The target protein is purified by affinity chromatography Ni-NTA,and pure SinH protein is obtained.The above research results provide basis for further exploration of soluble protein expression of Streptomyces in E.coli.