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

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• 作者：Carter ; Darrick ; Donald ; Robert G.K. ; Roos ; David ; Ullman ; Buddy
• 关键词：Uracil phosphoribosyltransferase ; Phosphoribosyltransferase ; Toxoplasma gondii ; Pyrimidine metabolism ; Protein purification ; Enzyme kinetics ; FUra ; 5-fluorouracil ; HGXPRT ; hypoxanthine-guanine-xanthine phosphoribosyltransferase ; OPRT ; orotate phosphoribos
• 刊名：Molecular and Biochemical Parasitology
• 出版年：1997
• 出版时间：August, 1997
• 年：1997
• 卷：87
• 期：2
• 页码：137-144
• 全文大小：287 K

文摘

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 μM for uracil and 243 μM for phosphoribosylpyrophosphate, respectively. Other naturally occurring pyrimidine or purine bases were not recognized as substrates. [¹⁴C]Uracil phosphoribosylation was inhibited by 5-fluorouracil with a K_i value of 25 μM 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.