Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii

Molecular and Biochemical Parasitology
Sonia M Robles LopezB H Zimmermann

Abstract

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 pI of 5.72, and was most similar to type IIa DHOases. A recombinant protein was expressed and purified with a yield of approximately 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 micromol/min/mg in the biosynthetic direction and 18.4 micromol/min/mg in the degradative direction, with K(m, carbamyl aspartate) = 323 microM and K(m, dihydroorotate) = 64.3 microM, 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.

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Citations

Dec 18, 2007·Biochemical and Biophysical Research Communications·Sudaratana R KrungkraiJerapan Krungkrai
Apr 7, 2018·Frontiers in Cellular and Infection Microbiology·Heidy Y Narvaez-OrtizBarbara H Zimmermann

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