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