小鼠金属硫蛋白-I在大肠杆菌中的表达与分离纯化

EXPRESSION OF MOUSE METALLOTHIONEIN I GENE IN ESCHERICHIA COLI AND PURIFICATION OF ITS PRODUCTS

目的 :在大肠杆菌中表达小鼠金属硫蛋白 I ,并研究其分子结构和生物学功能。方法 :将小鼠金属硫蛋白 I基因克隆入融合表达载体 pGEX 4T 1中 ,转化大肠杆菌BL2 1,得到含重组表达质粒 pGMA的工程菌。结果 :经IPTG诱导 ,表达出分子量约为 33KD的融合蛋白。然后经Glutathione Sepharose 4B亲和纯化后 ,用凝血酶切除GST部分。经SephacrylS 10 0过滤纯化 ,得到结合有Cd2 + 的小鼠金属硫蛋白 I。产率约为 3mg/L培养液。 结论 :成功制备了表达MT的工程菌。

Objective:To express mouse metallothionein I in Escherichia coli and study its molecular structure and function.Methods:Gene encoding the mouse MT I was cloned into fusion vector pGEX 4T 1 and transformed into Escherichia coli BL21.Results:With IPGT inducing,a fusion protein whose molecular weight was about 33KD was expressed.The fusion protein was purified through Glutathione Sepharose 4B affinitive chromatography and GST part was cut off with thrombin.Finally,the mixture was purified through Sephacryl S 100 gel filtration and the purified MT I protein binding Cd 2+ was obtained.The productivity is 3mg/L cultrure.Conclusion:The recombinant MT expression vector was successfully constructed.