EXPRESSION OF TRIMERIC HUMAN DUTP PYROPHOSPHATASE IN ESCHERICHIA-COLIAND PURIFICATION OF THE ENZYME

In order to rapidly purify human dUTPase, a cDNA fragment that encodes the enzyme was subcloned and expressed using the Escherichia coli pla smid vector pGEX2T. The resulting plasmid expressed high levels of a g lutathione S-transferase-dUTPase fusion protein following induction wi th IPTG. Affinity chromatography was used to purify the fusion protein , and dUTPase was then released from the fusion protein by thrombin tr eatment. The purified dUTPase has two additional vector-encoded residu es at the amino terminus (gly-ser), but they have no apparent effect o n the activity of the enzyme since the recombinant dUTPase has catalyt ic properties similar to those reported for dUTPase purified from huma n cells (32.3 U/mg, k(cat) = 25 s-1, K(m) = 2.6 muM). Enzyme activity was inhibited by 5-mercuri-dUTP and was shown to be sensitive to EDTA. Periodate-oxidized UTP had no effect on the activity of the enzyme, a nd dTTP caused only slight inhibition. The results of gel filtration e xperiments are consistent with a homotrimeric subunit composition for dUTPase. The ability to purify human dUTPase from E. coli should allow further characterization of the enzyme and provide material for the s creening of potentially useful inhibitors. (C) 1994 Academic Press, In c.