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Gene cloning, expression and purification of human mitochondrial tRNA~(Leu(UUR)) and its mutant |
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| Authors: | HAN Weiguo CHEN Li LIU JingZHAXiliang JIN Youxin WANG DebaoState Key Laboratory of Molecular Biology |
| Institution: | HAN Weiguo CHEN Li LIU JingZHAXiliang JIN Youxin WANG DebaoState Key Laboratory of Molecular Biology,Shanghai Institute of Biochemistry,Chinese Academy of Sciences,Shanghai 200031,China,Department of Biochemistry,Shanghai Medical University,Shanghai 200032,China |
| 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 repu-rified by 15% PAGE/urea. Their kinetic parameters for E.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). |
| Keywords: | human mitochondrial tRNALeu(UUR) mutation gene cloning expression purification |
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