Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant

Genes of human mitochondrial tRNA^Leu(UUR) (mtRNA^Leu(UUR)) and its mutant (mtRNA^Leu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively.E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNA^Leu(UUR) and mtRNA^Leu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters forE.coli LeuRS were measured. The results showed that the value of k_cal/ K_m of mtRNA^Leu(M) was about one fifth of that of mtRNA^Leu(UUR) and indicated the leucine acceptability of mtRNA^Leu(M) was much lower than that of mtRNA^Leu(UUR).