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The gene encoding carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) from Trichomonas vaginalis has been sequenced and its expression in this protozoon has been verified using reverse-transcription polymerase chain reaction. The codon usage and percentage nucleotide composition in the coding and noncoding regions are consistent with other genes isolated from this parasite. Phylogenetic analysis of this gene has suggested possible 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 using affinity chromatography. The expression of the recombinant protein was identified via Western blotting and matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides. Preliminary kinetic assays have revealed that the recombinant enzyme has a K(m) similar to that of the native enzyme and size-exclusion chromatography has shown that the enzyme is active as the homodimer.

The arginine dihydrolase pathway in Giardia intestinalis produces energy via the carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) reaction. Characterisation of the CBK gene from the Portland 1 strain indicated that it is located on either chromosome 3 or 4, does not appear to contain introns and is expressed in both the trophozoite and early cyst stages. Heterologous expression of CBK in Escherichia coli, using the pQE-30 expression system (QIAGEN), enabled a one-step purification of the recombinant enzyme via affinity chromatography. The expressed protein was identified by enzyme assay and mass spectrometry. The native and recombinant forms of the enzyme have similar physical properties and the recombinant enzyme appears to be active as the homodimer.

The gene encoding S-adenosylhomocysteinase activity (S-adenosylhomocysteine hydrolase, SAHH; EC 3.3.1.1) in Trichomonas vaginalis has been expressed in Escherichia coli to facilitate the characterisation of the enzyme. Expression of this gene using the pQE-30 (6xHis N-terminal tag) expression system (QIAGEN) has enabled the one-step purification of 6 mg of active recombinant enzyme from a 100-ml bacterial culture by affinity chromatography using a nickel-NTA matrix. The recombinant enzyme has a molecular weight of approximately 56,000 and identification of tryptic peptides by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry has shown that the purified recombinant protein is identical in primary structure to the predicted sequence. The presence of the N-terminal 6xHis tag in the recombinant enzyme did not appear to affect its kinetic and other properties, which are similar to those exhibited by the "native" enzyme present in cell-free extracts of T. vaginalis. These properties include a similar apparent Km for adenosine (20-25 microM for the recombinant and 5-10 microM for the native enzymes, respectively) and similar inhibition/inactivation patterns exhibited by adenosine analogues such as arabinosyl adenine (ara-A)., (Copyright 1998 Academic Press.)