新载体pET-DB对带有His-tag的仓鼠二氢叶酸还原酶的高效表达与纯化

High Level Soluble Expression and Purification of The His-tagged Chinese Hamster Dihydrofolate Reductase in E.coli Using a Newly Engineered pET-DB Vector

报道了带有His tag的仓鼠二氢叶酸还原酶基因的克隆和在DB序列增强下T7启动子调控该基因在大肠杆菌中的可溶性高效表达 ,SDS PAGE分析表明 ,带有His tag的仓鼠二氢叶酸还原酶的含量可占大肠杆菌细胞总蛋白质含量的 4 6 % .该酶的纯化可用常规的金属络合树脂一步纯化至SDS PAGE一条带 ,经凝血酶切去His tag的仓鼠二氢叶酸还原酶与用等电聚焦法获得的无His tag的酶有相同的酶活性 .

His-tagged Chinese hamster dihydrofolate reductase ( DHFR) expression in pET vector system has been reported very low. A newly modified pET protein expression vector, pET-DB, was utilized to overexpress of it in soluble form. The amount of DHFR reaches to 46% of the total protein in E. coli cells. This His-tagged DHFR could be purified routinely by Ni-NTA agarose resin and the His-tag could be removed by thrombin easily. This engineered DHFR has the same enzyme activity as the enzyme without His-tag obtained by iso-electrophoresis.