泡球蚴Em18重组蛋白在大肠杆菌中的表达和纯化方法的优化

Optimization of the prokaryotic expression and purification of pET41a-Em18 recombinant plasmid in Escherichia.coli

目的:筛选和优化泡球蚴Em18重组蛋白在大肠杆菌中表达和纯化的方法,为进一步筛选Em18抗原表位奠定基础。方法:原核表达pET41a-Em18重组质粒,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导、表达,获得rEm18-GST蛋白,采用不同的超声程序破碎大肠杆菌细胞,分别采用GST柱、His柱及两者结合方法,改变不同的平衡、洗涤和洗脱缓冲液浓度,纯化rEm18-GST蛋白。结果:(1)rEm18-GST蛋白在相对分子质量为50kDa处有表达条带,蛋白表达量随诱导时间的延长而增加;(2)超声程序为工作1s,间歇3s,每个循环时程10min,加入蛋白酶抑制剂,可降低目的蛋白的降解;(3)采用单纯His柱可获得回收量高、纯度高的rEm18-GST蛋白。结论:(1)对含有GST-tag和His-tag双重标签重组蛋白的纯化,应用His柱更有效,更经济;(2)建立和优化一套从大肠杆菌细胞中分离纯化重组Em18蛋白的有效方法。

Objective： To optimize the prokaryotic expression and purification of pET41a-Em18 in Escherichia. coli in order to be the basis for further screening for Em18 related peptides. Methods： The recombinant plasmid pET41a-Em18 was expressed with induction of IPTG. rEm18-GST proteins were broken up by the different procedure of sonication, rEm18-GST was purified by GST resin, His resin and both respectively, which concentration was changed in equilibration buffer, washing buffer and elution buffer. Results： （1） The rEm18-GST protein can be detected as a band of 50kDa and be improved with inducible time. （2） The high concentration of rEm18-GST was obtained by the optimized procedure of sonication： work Is, rest 3s. （3） High efficiency and purity protein were obtained by only the His resin. Conclusion： （1） It is more efficiency and economic to use His resin with optimized conditions to purify the recombinant protein containing both GSF-tag and His-tag. （2） One efficient method of isolating recombinant Em18 from E. coli was established.