Trichomonas vaginalis: Characterization, expression, and phylogenetic analysis of a carbamate kinase gene sequence

The gene encoding carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) from Trichomonas vaginalis has been sequenced and its expressio n in this protozoon has been verified using reverse-transcription polymeras e chain reaction. The codon usage and percentage nucleotide composition in the coding and noncoding regions are consistent with other genes isolated f rom this parasite. Phylogenetic analysis of this gene has suggested possibl e speciation events that are congruent with other studies, with suggestions of lateral gene transfer. The gene was expressed in Escherichia coli using the pQE-30 expression system, and the recombinant protein was purified usi ng affinity chromatography. The expression of the recombinant protein was i dentified via Western blotting and matrix-assisted laser desorption ionizat ion mass spectrometry of tryptic peptides. Preliminary kinetic assays have revealed that the recombinant enzyme has a K-m similar to that of the nativ e enzyme and size-exclusion chromatography has shown that the enzyme is act ive as the homodimer. (C) 2000 Academic Press.