A full-length cDNA for the mature, mitochondrial form of human mitochondrial RNA polymerase was cloned and expressed under the control of T5 or tac promoter in Escherichia coli, The cDNA was efficiently expressed at 37 degreesC, but virtually all the polymerase produced was insoluble, and renaturation of the inclusion bodies was unsuccessful. When the cells were grown at 25 degreesC, however, a portion of approximately 10% was soluble and active. The protein was purified 100-fold from the soluble lysates to homogeneity by two-step chromatography using Ni-nitrilotriacetic acid-Sepharose and heparin-agarose columns, as an N-terminal histidine tag attached and as the tag cleaved away. The purified polymerases with and without the histidine tag were both active in RNA polymerization in vitro as measured with poly(dA-dT) template, and specific activity was 140,000 units/mg. The purified enzyme has the same biochemical properties as the polymerase fraction partially purified from the human mitochondria, except for the promoter-specific activity that was not observed with the purified polymerase in the presence of mitochondrial transcription factor A. Additional factor(s) and/or mammalian-specific or regulatory modification(s) of the polymerase should be necessary for promoter-specific transcription. (C) 2001 Academic Press.