Purification of hyperexpressed Bacillus subtilis tRNA(Trp) cloned in Escherichia coli.
To study the effects of evolutionary sequence changes on the molecular interactions of tRNA inside the cell, the Bacillus subtilis tRNA(Trp) gene has been cloned into Escherichia coli JM109 under the control of the lac promotor. Hyperexpression of the gene in minimal medium upon induction yielded 28% of total tRNA in the form of B. subtilis tRNA(Trp). The tRNA(Trp) gene product was purified by the use of a single Vydac C4 high-performance liquid chromatography (HPLC) matrix. This experimental system provided a valuable system for the hyperexpression and purification of a heterologous tRNA for studies in vitro. Moreover, because HPLC fractionation of the heterologous tRNA(Trp) gene product yielded multiple peaks, the system made possible an analysis of the molecular mechanisms for the transcriptional modifications of the tRNA(Trp) gene product in vivo.
Department of Biochemistry, University of Toronto, Ontario, Canada.
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D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007763:Lac Operon; D008969:Molecular Sequence Data; D010957:Plasmids; D012364:RNA, Transfer, Trp / Q000096:biosynthesis / Q000302:isolation & purification*
D012364:RNA, Transfer, Trp