教育部留学回国人员科研启动基金(No.039)和中国科学院生物物理研究所所长基金资助项目(No.25).
This work was supported by Ministry of Education of China for returned overseas Chinese scholars (039) and Institute of Biophysics, The Chinese Academy of Sciences (025).
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46%.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.
朱志勇,谭波,张文河,张洪杰.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[J].生物化学与生物物理进展,2004,31(7):655-658
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