Abstract
A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA(1)(Leu)(CAG) and Aquifex aeolicus tRNA(Leu)(GAG) with higher catalytic activity in the leucylation of E. coli tRNA(Leu) than that previously expressed in E. coli without the N-terminal 21 residues.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Cells, Cultured
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Cloning, Molecular
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Histidine / genetics
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Humans
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Lepidoptera / cytology
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Lepidoptera / genetics
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Leucine-tRNA Ligase / genetics
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Leucine-tRNA Ligase / isolation & purification
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Leucine-tRNA Ligase / metabolism*
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Mitochondria / genetics
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Mitochondria / metabolism*
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Molecular Sequence Data
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Peptide Fragments / metabolism
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Protein Engineering / methods*
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Protein Precursors / metabolism
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Protein Transport
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RNA, Transfer, Leu / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Spectrometry, Fluorescence
Substances
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Escherichia coli Proteins
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Peptide Fragments
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Protein Precursors
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RNA, Transfer, Leu
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Recombinant Proteins
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Histidine
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Leucine-tRNA Ligase