突变型大肠杆菌酒石酸脱氢酶β亚基基因的克隆表达与包涵体的复性

CLONING OVEREXPRESSION AND REFOLDING OF MUTANT ESCHERICHIA COLI TARTRATE DEHYDRATASE BETA SUBUNIT

目的利用生物工程相关技术,获得不含Cys的突变型大肠杆菌酒石酸脱氢酶β亚基,为下一步蛋白质交联奠定基础。方法利用PCR定点突变技术扩增目的基因,并将之克隆到质粒PGM-T,转化大肠杆菌DH5α。经测序证明序列无误后,将之与表达载体pTrcHisC连接,在大肠杆菌BL21中经IPTG诱导表达。目的蛋白用TALON金属亲和树脂纯化,通过分步透析逐步去除变性剂的方法复性。结果通过SDS-PAGE分析,确定目的蛋白以包涵体形式存在。复性产率可达70%。结论此次实验所得到的目的蛋白的纯度与活性均满足蛋白质交联的需要。

Objective To obtain the mutant Tartrate Dehydratase Beta Subunit so as to reseach protein crosslinking. Methods By using the Polymerase Chain Reaction（PCR） point mutation technique, the mutant TtdB was amplified. The fragment was inserted into plasmid PGM-T by the sites of two restriction enzyme BamHⅠ and Hind Ⅲ, and the target gene was confirmed by DNA sequencing. Then the target gene was subcloned into the expression plasmid pTrcHisC. The recombinant protein expression was induced by IPTG in Escherichia coli BL21. The recombinant protein was purified by immobilized metal affinity chromatography（IMAC）. Results The expression was associated with formation of inclusion bodies with SDS-PAGE electrophoresis and refolded by dialysis which resulted in a recovery rate exceeding 70 %. Conclusion Purity and activity of the mutant target protein satisfy next research on protein cross-linking.