Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli.
The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase. The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons. The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence. A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E. C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases. Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction. Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band. The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield. The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.
Weiner
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Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany.
Weiner
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Maley
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Maley
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GF
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003844:DCMP Deaminase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004247:DNA; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA; D004247:DNA; D003844:DCMP Deaminase