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

Darrick Carter; Robert G.K. Donald; David Roos; Buddy Ullman

doi:10.1016/S0166-6851(97)00058-3

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

Darrick Carter
, Robert G.K. Donald
, David Roos
, Buddy Ullman

Biochemistry & Molecular Biology

Research output: Contribution to journal › Article › peer-review

37 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	137-144
Number of pages	8
Journal	Molecular and Biochemical Parasitology
Volume	87
Issue number	2
DOIs	https://doi.org/10.1016/S0166-6851(97)00058-3
State	Published - 1997

Funding

This work was supported by Grant AI-31808 (D.S.R. and B.U.) from the National Institute of Allergy and Infectious Disease. D.C. was supported in part by an N.L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon. D.S.R. and B.U. are both Burroughs Wellcome Fund Scholars in Molecular Parasitology, and this work was supported in part by grants from the Burroughs Wellcome Fund.

Funders	Funder number
Oregon Medical Research Foundation
National Institute of Allergy and Infectious Diseases	U01AI031808
Burroughs Wellcome Fund

Keywords

Enzyme kinetics
Phosphoribosyltransferase
Protein purification
Pyrimidine metabolism
Toxoplasma gondii
Uracil phosphoribosyltransferase

ASJC Scopus subject areas

Parasitology
Molecular Biology

Access to Document

10.1016/S0166-6851(97)00058-3

Cite this

@article{97ba19a3e52e4423967a0c41d8077258,

title = "Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii",

abstract = "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.",

keywords = "Enzyme kinetics, Phosphoribosyltransferase, Protein purification, Pyrimidine metabolism, Toxoplasma gondii, Uracil phosphoribosyltransferase",

author = "Darrick Carter and Donald, \{Robert G.K.\} and David Roos and Buddy Ullman",

note = "Funding Information: This work was supported by Grant AI-31808 (D.S.R. and B.U.) from the National Institute of Allergy and Infectious Disease. D.C. was supported in part by an N.L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon. D.S.R. and B.U. are both Burroughs Wellcome Fund Scholars in Molecular Parasitology, and this work was supported in part by grants from the Burroughs Wellcome Fund. ",

year = "1997",

doi = "10.1016/S0166-6851(97)00058-3",

language = "English (US)",

volume = "87",

pages = "137--144",

journal = "Molecular and Biochemical Parasitology",

issn = "0166-6851",

publisher = "Elsevier B.V.",

number = "2",

}

TY - JOUR

T1 - Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii

AU - Carter, Darrick

AU - Donald, Robert G.K.

AU - Roos, David

AU - Ullman, Buddy

N1 - Funding Information: This work was supported by Grant AI-31808 (D.S.R. and B.U.) from the National Institute of Allergy and Infectious Disease. D.C. was supported in part by an N.L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon. D.S.R. and B.U. are both Burroughs Wellcome Fund Scholars in Molecular Parasitology, and this work was supported in part by grants from the Burroughs Wellcome Fund.

PY - 1997

Y1 - 1997

N2 - 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.

AB - 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.

KW - Enzyme kinetics

KW - Phosphoribosyltransferase

KW - Protein purification

KW - Pyrimidine metabolism

KW - Toxoplasma gondii

KW - Uracil phosphoribosyltransferase

UR - https://www.scopus.com/pages/publications/0030738303

UR - https://www.scopus.com/pages/publications/0030738303#tab=citedBy

U2 - 10.1016/S0166-6851(97)00058-3

DO - 10.1016/S0166-6851(97)00058-3

M3 - Article

C2 - 9247925

AN - SCOPUS:0030738303

SN - 0166-6851

VL - 87

SP - 137

EP - 144

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

IS - 2

ER -

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

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this