Expression in Escherichia coli 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.
D000818:Animals; D004247:DNA / Q000032:analysis; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009876:Operon; D009954:Ornithine Carbamoyltransferase / Q000096:biosynthesis*; D010957:Plasmids; D014176:Protein Biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D004247:DNA; D009954:Ornithine Carbamoyltransferase