大肠杆菌T蛋白的高效表达和分离纯化

High efficient expression and purification of the Escherichia coli T-protein

目的 构建高效表达菌株 ,以大量表达大肠杆菌T蛋白 ,为后续的新药作用靶点研究提供活性蛋白质材料。方法 利用高效表达质粒pET2 6b+ 克隆了大肠杆菌TyrA基因 ,并在其C端融合了一个由 6个组氨酸组成的his tag以利分离纯化。 结果 T蛋白的表达量达每 1L培养液 2 0 0mg ,细胞裂解液经一次his tag亲和柱色谱 ,纯度达 98% ,分支酸变位酶和预苯酸脱氢酶的比活性分别为 130和 98U·mg-1。结论 本法表达的大肠杆菌T蛋白 ,表达量高 ,纯化容易 ,酶活性正常 ,为进一步研究T蛋白的结构与功能 ,并进行新药设计奠定了基础。

OBJECTIVE: To clone and express the bi-functional T-protein to provide active protein as a target for new drug development. METHOD: The pEq26b + vector was employed in the cloning of TyrA gene, and a his-tag was fused in the C-terminals of T-protein to facilitate purification. RESULTS: The amount of T-protein expressed exceeded 200 mg per liter. The purity of T-protein was higher than 98% after one step his-tag affinity chromatography of the lysate. The specific activities of chorismate mutase and prephenate dehydrogenase were 130 and 98 U&middotmg-1 respectively. CONCLUSION: T-protein expressed in this study was high, easy to purify and normal in the enzyme activity.