Abstract
Orotidine-5'-monophosphate decarboxylase (OD-Case) catalyzes the conversion of orotidine 5'-monophosphate to UMP. In mammals, ODCase is present as part of a bifunctional protein which also contains orotate phosphoribosyltransferase; the preceding enzyme in the de novo UMP biosynthetic pathway. We have isolated a plasmid (pMEJ) which contains a cDNA for the ODCase domain of UMP synthase. Insertion of this sequence into an Escherichia coli expression vector (pUC12) has allowed for the expression of ODCase and not orotate phosphoribosyltransferase in E. coli. The molecular weight of the expressed protein is 26,000-27,300 from immunoblot analysis which corresponds closely to the molecular weight of the ODCase domain (28,500) isolated by tryptic digestion of UMP synthase. We have sequenced the cDNA insert of pMEJ and deduced the amino acid sequence. The molecular weight of the ODCase domain calculated from the amino acid sequence in 28,654. Comparison of the deduced amino acid sequence from pMEJ with that for yeast ODCase (a monofunctional protein) demonstrated that 52% of the amino acids were identical when the two sequences are compared. Furthermore, several stretches of the amino acid sequence have 80% or greater absolute homology.
Highlights
From the Department of Bi0chemistr.y and Nutrition,School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514
We report the expression in E. coli of the ODCase domain of Ehrlich ascites UMP synthase
We have reported the isolation of a cDNA clone, pMEJ, from an Ehrlich ascites cDNA library which contains a nucleotide coding sequence for a portion of the mRNA of UMP synthase
Summary
Plasmid, pMEJ-E, was digested with EcoRI and the 1600-bp EcoRI fragment was isolated and insertedin both orientations. Transformants expressing functional OPRTase or ODCase donot require added that pMEJcontained coding sequences for UMP synthase, a portion of the insert was subcloned into theexpression vector pUC12 [29]. The E. coli ODCase did not cross-react with UMP synthase-specific serum Selection for plasmid-containing cells was per- synthase inthe ammonium sulfate fraction of Ehrlich ascites formed by plating on minimal medium with ampicillin (25 pg/ml) extracts [2]. Lane 5 contains extractsfrom pUC12-transformed AT2538 cells which contain active E. coli ODCase. The calculatedmolecular weight of the proteinexpressed from pODC is 28,200, including the translatedsequences from the lac Z gene and the EcoRI linker This is similar to the size determined for the expressed domain (26,000 to 27,300) by immunoblotting (Fig. 4).These resultssuggest that theregion beyondnucleotide 799 does not contain protein coding sequences (see “Discussion”).
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