Expession And Purification Of Glutaminyl-tRNA Synthetase

Aminoacyl-tRNA synthetase( AARS) is a kind of key enzymes in the process of protein synthesis. Glutamine-tRNA synthase(GlnRS) has anti-apoptotic effect with dependent on glutamine in tumor cells. Therefore, researches on its structure, function and stability of GlnRS has the great significance. Human GlnRS was cloned first, and then the expression vector pET-28a-GlnRS was constructed, and it was transformed into E.coli BL21(DE3). The results showed that the protein expression could attain the largest amount when the E.coli BL21(DE3) was induced by 0.33 mM IPTG and at the tempreture of 16℃ overnight. Ultrasonic disruption cells were collected and protein crude extracts was obtained. By using Ni-NTA agarose and Q-column finally seperetedly, the protein crude extracts was purified and target protein was gotten finally, which was tested by gel electrophoresis as a single band. The activity of GlnRS was detected by using isotopic methods, and the results showed that the glutamine had no inhibition substrate.Meanwhile, The expression vector of Arginine-t RNA synthase(ArgRS) was constructed in order to coexpression of human GlnRS and ArgRS. The soluble expression of GlnRS in E.coli was improved by increasing the stability of GlnRS. In addition, a virus vector Bacmid-GlnRS were constructed, then GlnRS was expressed successfully, and finally GlnRS was obtained and tis molecular weight was slightly higher than the theoretical molecular weight. In this experiment, Glutamine-tRNA synthase() was successfully expressed in prokaryotic and eukaryotic cells and the relationship between the enzyme and substrate concentration was analysed as well. These results are important to explore GlnRS nonclassical feature, and to lay the preliminary groundwork for future crystal crystallization.