Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant |
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Authors: | Weiguo Han Li Chen Jing Liu Xiliang Zha Youxin Jin Debao Wang |
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Institution: | (1) State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 200031 Shanghai, China;(2) Department of Biochemistry, Shanghai Medical University, 200032 Shanghai, China |
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Abstract: | Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and its mutant (mtRNALeu(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 mtRNALeu(UUR) and mtRNALeu(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 kcal/ Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR). |
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Keywords: | human mitochondrial tRNALeu(UUR) mutation gene cloning expression purification |
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