A Novel System for Hyper Expression and
Rapid Purification of Arginyl-tRNA Synthetase from Escherichia coli
LIU Wen, WANG
En-Duo* and WANG Ying-Lai
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China
)
Abstract 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.
Key Words E.coli； arginyl-tRNA synthetase；
overexpression； purification
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