Recombinant human cytidine deaminase: expression, purification, and characterization.
The complementary DNA (cDNA) coding for human cytidine deaminase (CDA) was obtained using two specific primers to screen RNA from peripheral blood polymorphonuclear leukocytes by reverse transcriptase PCR. The cDNA fragment was ligated into the expression vector pTrc99-A and expressed in Escherichia coli following induction with isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The nucleotide sequence of the cDNA corresponded to that published by Laliberté and Momparler (Cancer Res. 54, 5401-5407, 1994). It contained a 438-bp open reading frame encoding a polypeptide of 146 amino acids with a predicted molecular mass of 16.2 kDa. The protein expressed in E. coli showed high cytidine deaminase activity and its molecular mass was estimated to be 57 kDa by gel filtration and 16 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both values are in agreement with those already published and suggest that human CDA contains three or four identical subunits. Cross-linking experiment indicated that the enzyme is a tetramer. The recombinant CDA has been purified to homogeneity by a rapid procedure consisting of heat inactivation followed by affinity chromatography. The final enzyme preparation showed a specific activity of 105 U/mg, corresponding to about 88-fold purification with respect to the crude extract and was judged to be >98% pure by SDS-PAGE. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis revealed the presence of 1 atom of Zn per subunit. Since CDA causes the deamination of several antitumoral cytidine-analog drugs, the recombinant enzyme was characterized kinetically and several pyrimidine nucleoside analogs were tested as potential substrates and inhibitors. The results obtained agreed closely with those previously reported for the purified human placenta CDA.
Vincenzetti
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Dipartimento di Biologia M.C.A., Università di Camerino, via Camerini, 3, Camerino (MC), 62032, Italy.
Cambi
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Neuhard
J
J
Garattini
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E
Vita
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003564:Cytidine Deaminase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D003564:Cytidine Deaminase; D015032:Zinc