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 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.
Minotto
L
L
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, New South Wales, 2052, Australia.
Edwards
M R
MR
Bagnara
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016384:Consensus Sequence; D016054:DNA, Protozoan / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008969:Molecular Sequence Data; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014246:Trichomonas vaginalis / Q000145:classification / Q000201:enzymology / Q000235:genetics*
D016054:DNA, Protozoan; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase