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 li gated 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 conventiona l protein purification techniques. In solution, UPRT behaved as a mono mer and exhibited K-m(app) values of 3.5 mu M for uracil and 243 mu M for phosphoribosylpyrophosphate, respectively. Other naturally occurri ng 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 r ecombinant enzyme are now available for biochemical and structural stu dies, facilitating evaluation of UPRT as a possible therapeutic target . (C) 1997 Elsevier Science B.V.