Overproduction and purification ofEscherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA1Leu and tRNA2Leu 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 tRNA1Leu was 50% of total tRNA from MT-Leu1, while that of tRNA2Leu 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.

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