Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli Molecular cloning of rat ornithine transcarbamylase. Molecular cloning of rat ornithine transcarbamylase

Abstract

A complementary DNA [cDNA] clone, corresponding to the mature form of ornithine transcarbamylase (OTCase), was selected from a rat liver cDNA library constructed in bacteriophage .lambda.gt10. OTCase clones were selected using a synthetic DNA probe of 15 bases, corresponding to the 3'' end of the OTCase mRNA. Putative OTCase clones were subcloned into the expression vector, pUC9. The identity of inserts was confirmed by colony immunoassay and by electrophoretic transfer of cloned proteins from sodium dodecyl sulfate/polyacrylamide gels to nitrocellulose filters, followed by probing with monospecific anti-OTCase antibodies and 125I-labeled protein A. A clone corresponding to the full-length mature form of rat liver OTCase (plus 15 amino acids from E. coli .beta.-galactosidase) was obtained, and the identity of the clone was confirmed by comparison of the 5'' sequence with a limited N-terminal amino acid sequence. A sequence discrepancy, between the published sequence and the sequence predicted from the cDNA structure, is noted.