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