Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter.
Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter.
复制标题
大肠杆菌中 5-烯醇丙酮莽草酸-3-磷酸合酶的过量生产：T7 启动子的使用。
DOI：
10.1093/protein/5.5.461
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Evans,JN
中科院分区：
文献类型：
--
作者：
Shuttleworth,WA;Hough,CD;Bertrand,KP;Evans,JN
大肠杆菌aroA基因的产物5-烯醇式莽草酸-3-磷酸（EPSP）合成酶在大肠杆菌BL 21（λ DE 3）中，在T7基因10启动子和核糖体结合位点的控制下，可过量表达至细胞总蛋白的约50%。EPSP合成酶是芽后除草剂草甘膦（通常称为RoundupTM）的主要靶标。描述了一种简单的两步纯化，其导致99%纯的均质蛋白（如通过PAGE测定的）。通过测定其动力学参数、N-末端蛋白质和DNA序列、氨基酸分析和13 C-NMR光谱，将该蛋白质的完整性与先前表征的来自大肠杆菌AB 2829（pKD 501）的蛋白质进行了比较。这种新的高产菌株很容易提供的活性位点的NMR研究和开发新的时间分辨固态NMR技术目前正在本实验室所需的高纯度蛋白质的克数量。
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the product of theEscherichia coli aroAgene, has been overproduced inE.coliBL21(λDE3) under the control of the T7 gene10promoter and ribosome binding site, to a level of ˜50% of total cell protein. EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as RoundupTM. A simple two step purification is described, which results in 99% pure homogeneous protein (as determined by PAGE). The integrity of the protein has been compared with previously characterized protein from.E.coliAB2829(pKD501) by determination of its kinetic parameters, N-terminal protein and DNA sequences, amino acid analysis and13C-NMR spectroscopy. This new overproducing strain readily provides the gram quantities of highly pure protein required for NMR studies of the active site and the development of novel time-resolved solid-state NMR techniques currently underway in this laboratory.