Effect of Deletion of 245 and 252 Arginines in E. coli Arginyl-tRNA Synthetase on Its Structure and Function.
In order to study structure and function of E.coli arginyl-tRNA synthetase(ArgRS), deletion of R245 and 252 in this enzyme was carried out by site-directed mutagenesis of gene coding for this enzyme, respectively. The genes, argSdeltar245 and argSdeltar252-encoding ArgRS mutant, ArgRSdeltaR245 and ArgRSdeltaR252 were obtained and cloned into pUC18 or pTrc99B, respectively. The expression of argSdeltar245 and the properties of the mutant ArgRSdeltaR245 were studied. The gene encoding ArgRS, argS was expressed as a soluble protein; however, ArgRS deleted R245 formed inclusion body during the expression of argSdeltar245 in E. coli. After dissolution of the inclusion body by 8 mol/L urea or 7 mol/L guandidine chloride, followed by renaturation by the methods of gel filtration or dilution, the specific activity of the mutant enzyme, ArgRSdeltaR245 was about 40 unit/mg, that is only 0.3% activity of the native enzyme. By detection of ultraviolet absorbance and difference spectroscopy and titration of DTNB, the conformations of the native and mutant enzymes were compared. It was shown that the tertiary structure of ArgRSdeltaR245 was more relaxed than that of ArgRS, and the chromophoric groups in the mutant enzyme were more exposed than that of the native enzyme. It may be the main reason of forming inclusion body during the mutant gene expression and losing activity of enzyme.
Wu
Ji-Fu
JF
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