Study on the Overexpression of the Gene Encoding Arginyl-tRNA Synthetase under Induction.
Arginyl-tRNA synthetase (ArgRS) from E. coli was overproduced from transformant containing the gene encoding this enzyme (argS) by 550 fold higher than that from host cell. By site-directed mutagenesis the cleavage site for NcoI restriction endonuclease was introduced into the start codon of argS. The mutant gene was recombinated with plasmid pTrc99B under IPTG control. In the transformant containing the recombinated plasmid, argS can overexpressed 2 000 times higher than that in host cell. Through one step DEAE-Sepharose column chromatography, ArgRS was purified to be one band by SDS-PAGE and its specific activity was 15 000 u/mg, similar to reported values. The mutant ArgRS2ND which had the second residue asparagine replaced by aspartic acid showed no change of activity, kinetic constants nor the thermal and denaturational stabilities.
Wu
Jin-Fu
JF
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China.
Xia
Xian
X
Wang
En-Duo
ED
Wang
Ying-Lai
YL