High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli

Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
W ShiJ T Wong

Abstract

The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid. Upon induction with isopropyl-beta-D-thiogalactopyranoside, Escherichia coli JM109[pKSW1] cells synthesized TrpRS to a level corresponding to 45% of total cell proteins. This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by [3H]Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization. Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E. coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be deductible on the basis of complementation testing.

Citations

Sep 6, 2003·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Kuiyi Xing, Marjorie F Lou
Jul 29, 2004·The Journal of Biological Chemistry·Jie JiaYou-Xin Jin

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