Human mitochondrial leucyl-tRNA synthetase with high activity produced from Escherichia coli.
The processing of human mitochondrial leucyl-tRNA synthetase had been previously investigated in insect cell. In the present work, the gene encoding human mitochondrial leucyl-tRNA synthetase with the same N-terminus as that processed in the mitochondria of insect cell was cloned and expressed in Escherichia coli. The enzyme was purified by affinity chromatography on Ni-NTA column. About 6 mg of human mitochondrial leucyl-tRNA synthetase was obtained from 1 liter of culture. The specific activity of the purified enzyme is 127.7 units/mg, the highest activity of the reported results; this enzyme has the potential for characterizing the mitochondrial tRNA mutants associated with some human mitochondrion-related neuromuscular disorders. The kinetic constants for three substrates: leucine, ATP, and E. coli tRNA1Leu (CAG) in the leucylation reaction are also reported herein.
Yao
Yong-Neng
YN
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China.
Wang
Lie
L
Wu
Xiang-Fu
XF
Wang
En-Duo
ED
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D006801:Humans; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D007935:Leucine-tRNA Ligase