Objective: To construct the recombinant expression vector of human mitochondrial leucyl-tRNA synthetase(mtLeuRS),and to express, purify mtLeuRS and determine its kinetic parameters.Methods: The human mtLeuRS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-mtLeuRS,which could direct the synthesis of a mammalian mitochondrial derived protein in E. coli BL21-CodonPlus(DE3)-RIL.The vector allowed production and single-step purification of His6-tagged human mtLeuRS by the facilitation of metal (Ni2+) chelate affinity chromatography.As the substrate of mtLeuRS,the human mitochondrial tRNALeu(UUR) was synthesized and transcribed in vitro with T7 RNA polymerase.The ability of mtLeuRS to aminoacylate mitochondrial tRNALeu(UUR) was assayed by enzymatic reaction when tRNALeu(UUR) between 5-25 μmol/L.Results: The expression level of human mtLeuRS was about 1％-2％ of total cell proteins after isopropyl β-D-thiogalactoside induction.The produced human mtLeuRS-His6 could be purified to homogeneity within 2 h and about 2 mg purified enzyme could be obtained from 1 L cell culture.The procedure of purification was easy and simple.The mitochondrial tRNALeu(UUR) obtained was as high as 100 times of transcription template.The enzymatic reaction showed that the Km of mtLeuRS for tRNALeu(UUR) was 16.67 μmol/L and the turnover (Kcat) was 0.17 s-1. Conclusion: The mature form of human mitochondrial LeuRS has been cloned and expressed in E.coli.It is capable of aminoacylating tRNALeu(UUR) transcripted in vitro.