Expression in Escherichiacoli of functional precursor to the rat liver mitochondrial enzyme, ornithine carbamyl transferase. Precursor import and processing in vitro

cDNA encoding the full-length cytosolic precursor of rat liver ornithine carbamyl transferase, a mitochondrial matrix enzyme, was inserted into pKK223-3 and expressed under the control of the tac promoter. Following transformation of E. coli strain JM105 and induction by isopropylthiogalactoside, a polypeptide was synthesized which reacted with antibody against ornithine carbamyl transferase and co-migrated in SDS-polyacrylamide gels with authentic enzyme precursor (pOCT, Mr = 40,000 kDa); it constituted approximately 0.1% of total E. coli protein. pOCT was synthesized in vitro by coupled transcription-translation of the recombinant plasmid in an E. coli S30 extract; upon subsequent addition of purified rat liver or heart mitochondria, the precursor was imported and processed to mature form. Synthesis of pOCT in a bacterial system, therefore, results in production of a functional precursor polypeptide which does not require additional cytosolic factors from eukaryotic cells to support its uptake and processing by mitochondria in vitro.

Expression in Escherichiacoli of functional precursor to the rat liver mitochondrial enzyme, ornithine carbamyl transferase. Precursor import and processing in vitro Journal Articles