Overproduction and purification of Escherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA 1 Leu and tRNA 2 Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A260, respectively: the content of tRNA 1 Leu was 50% of total tRNA from MT-Leu1, while that of tRNA 2 Leu was 30% of total tRNA from MT-Leu2. Both tRNALeus from their rotal tRNs were fractionated to 1 600 pmol/A260 after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNALeu catalyzed by leucyl-tRNA synthetase were determined. Project supported by the National Natural Science Foundation of China (Grant No. 39570164).