Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii

The coding region derived from a full-length CDNA spanning the uracil phosphoribosyltransferase (UPRT) gene of Toxoplasma gondii has been ligated into a bacterial expression vector and overexpressed in E. coli. Recombinant UPRT protein migrated with a molecular mass of 27 kDa on SDS polyacrylamide gels and was purified to homogeneity by conventional protein purification techniques. In solution, UPRT behaved as a monomer and exhibited K-m(app) values of 3.5 mu M for uracil and 243 mu M for phosphoribosylpyrophosphate, respectively. Other naturally occurring pyrimidine or purine bases were not recognized as substrates, [C-14]Uracil phosphoribosylation was inhibited by 5-fluorouracil with a K-i value of 25 mu M and was not activated by GTP. Ample quanitities of recombinant enzyme are now available for biochemical and structural studies, facilitating evaluation of UPRT as a possible therapeutic target. (C) 1997 Elsevier Science B.V.