| 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 |
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| 引用本文： | 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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| 作者姓名： | 朱志勇 谭波 张文河 张洪杰 |
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| 作者单位： | 1. 北京师范大学生物化学与分子生物学系,北京,100875;中国科学院生物物理研究所,北京,100101
2. 中国科学院生物物理研究所,北京,100101 |
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| 基金项目： | 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). |
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| 摘 要： | 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.
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| 关 键 词： | 蛋白质表达，二氢叶酸还原酶，pET-DB载体，His-tag |
| 收稿时间： | 2004/1/12 0:00:00 |
| 修稿时间： | 2004/2/25 0:00:00 |
High Level Soluble Expression and Purification of The His-tagged Chinese Hamster Dihydrofolate Reductase in E.coli Using a Newly Engineered pET-DB Vector |
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| ZHU Zhi-Yong,TAN Bo,ZHANG Wen-He and ZHANG Hong-Jie.High Level Soluble Expression and Purification of The His-tagged Chinese Hamster Dihydrofolate Reductase in E.coli Using a Newly Engineered pET-DB Vector[J].Progress In Biochemistry and Biophysics,2004,31(7):655-658. |
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| Authors: | ZHU Zhi-Yong TAN Bo ZHANG Wen-He and ZHANG Hong-Jie |
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| Institution: | Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, China; Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China;Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, China; Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China;Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China;Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China |
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| Abstract: | 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. |
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| Keywords: | protein expression dihydrofolate reductase pET-DB vector His-tag |
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