一个高效表达、快速纯化大肠杆菌精氨酰-tRNA合成酶的新系统(英文)

编码大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶（ Ａｒｇ Ｒ Ｓ） 的基因ａｒｇ Ｓ 被克隆到ｐ Ｍ Ｆ Ｔ７５ 载体上。将此质粒转化的大肠杆菌 Ｊ Ｍ１０９（ Ｄ Ｅ３） 中， 该转化子粗抽液的比活是宿主菌的２ ５００ 倍。通过 Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ Ｆａｓｔ Ｆｌｏｗ 和 Ｂｌｕｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ两步柱层析在一天内即可将精氨酰ｔ Ｒ Ｎ Ａ 合成酶纯化至电泳一条带， 比活为３６ ０００ ｕ／ｍｇ ， 总收率可达６９ ％ 。与以前报道的 Ａｒｇ Ｒ Ｓ的高表达质粒相比， 使用该重组质粒可以很方便地将昂贵的标记氨基酸高效地参入酶分子内。目前的研究结果表明，该新系统能够很方便地提供大量的更高比活的大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶以进行该酶的 Ｎ Ｍ Ｒ 和结晶学研究

A Novel System for Hyper Expression and Rapid Purification of Arginyl tRNA Synthetase from Escherichia coli *

The gene argS encoding arginyl tRNA synthetase (ArgRS) in Escherichia coli has been cloned into the vector pMFT7 5 which allows overproduction of ArgRS. The specific activity of the synthetase in the crude extract of E.coli JM109(DE3) transformant containing the plasmid pMFT7 argS was approximately 2 500 times that of JM109(DE3) (the host strain without the vector). The overproduced synthetase can be purified to homogeneity with the specific activity of 36 000 units/mg under native condition within one day by a two step purification system of DEAE Sepharose CL 6B Fast Flow and Blue Sepharose CL 6B chromatographies with an overall yield of 69%. Moreover, this system makes it very easy to incorporate efficiently the expensive labeled amino acids into this enzyme. This novel system can conveniently provide a large amount of purified enzyme with a much higher specific activity than before which may be beneficial to NMR and crystallographic studies on this enzyme.