Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii

A full-length dihydroorotase (DHOase) sequence was cloned from a Toxoplasma gondii tachyzoite cDNA library. The sequence had a calculated molecular mass of 44.2 kDa and a p I of 5.72, and was most similar to type IIa DHOases. A recombinant protein was expressed and purified with a yield of ∼20 mg L...

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Bibliographic Details
Published in:Molecular and biochemical parasitology 2006-07, Vol.148 (1), p.93-98
Main Authors: Lopez, Sonia M. Robles, Triana, Miryam Andrea Hortua, Zimmermann, Barbara H.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Apicomplexa
Dihydroorotase
Dihydroorotase - chemistry
Dihydroorotase - genetics
Dihydroorotase - metabolism
Dihydroorotase - pharmacokinetics
Genes, Protozoan
Molecular Sequence Data
Molecular Weight
Protozoan Proteins - chemistry
Protozoan Proteins - genetics
Protozoan Proteins - metabolism
Protozoan Proteins - pharmacokinetics
Pyrimidine biosynthesis
Sequence Alignment
Toxoplasma - enzymology
Toxoplasma - genetics
Toxoplasma gondii
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Summary:A full-length dihydroorotase (DHOase) sequence was cloned from a Toxoplasma gondii tachyzoite cDNA library. The sequence had a calculated molecular mass of 44.2 kDa and a p I of 5.72, and was most similar to type IIa DHOases. A recombinant protein was expressed and purified with a yield of ∼20 mg L −1 of cell culture. Polyclonal antibodies raised against purified recombinant protein reacted with a band of the expected molecular mass in tachyzoite extracts. Specific activities of 18.3 μmol/min/mg in the biosynthetic direction and 18.4 μmol/min/mg in the degradative direction, with K m, carbamyl aspartate = 323 μM and K m, dihydroorotate = 64.3 μM, were measured for purified recombinant protein. Size exclusion chromatography/laser light scattering showed a single, monodisperse peak with a molecular mass of 45.6 kDa, suggesting that the native protein is a monomer.
ISSN:0166-6851
1872-9428
DOI:10.1016/j.molbiopara.2006.03.003