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Gene cloning, expression and purification of human mitochondrial tRNA(Leu(UUR)) and its mutant
Abstract:
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)).
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