OVERPRODUCTION AND PURIFICATION OF ESCHERICHIA-COLI TRNA(LEU)

Chemically synthesized genes encoding Escherichia coli tRNA(1)(Leu) an d rRNA(2)(Leu) were ligated into the plasmid pTrc99B, then transformed into Escherichia 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. A s a result, leucine accepting activity of their total tRNA reached 810 and 560 pmol/A(260), respectively: the content of tRNA(1)(Leu) was 50 % of total tRNA from MT-Leu1, while that of tRNA(2)(Leu) was 30% of to tal tRNA from MT-Leu2. Both tRNA(Leu)s from their total tRNAs were fra ctionated to 1 600 pmol/A(260) after DEAE-Sepharose and ED-cellulose c olumn chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of rRNA(Leu) catalyzed by leucyl-tRNA synthet ase were determined.