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.
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. wed@server.shcnc.ac.cn
Wen
W
Liu
En-Duo
ED
Wang
Ying-Lai
YL
Wang