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

Weiguo Han, Li Chen, Jing Liu, Xiliang Zha, Youxin Jin, Debao Wang

Research output: Contribution to journalArticle

3 Scopus citations

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 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).

Original languageEnglish (US)
Pages (from-to)113-120
Number of pages8
JournalScience in China, Series C: Life Sciences
Volume44
Issue number2
DOIs
StatePublished - Apr 2001

Keywords

  • Expression
  • Gene cloning
  • Human mitochondrial tRNA
  • Mutation
  • Purification

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Environmental Science(all)
  • Agricultural and Biological Sciences(all)

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