Trichomonas vaginalis: Characterization, Expression, and Phylogenetic Analysis of a Carbamate Kinase Gene Sequence

• Published in: Experimental parasitology Vol. 95; no. 1; pp. 54 - 62
• Main Authors: Minotto, Linda, Edwards, Michael R., Bagnara, Aldo S.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.05.2000; Elsevier
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Blotting, Western; carbamate kinase (CBK; carbamoyl phosphate synthetase (CPS); Consensus Sequence; dithiothreitol (DTT); DNA, Protozoan - chemistry; EC 2.7.2.2; Electrophoresis, Polyacrylamide Gel; ethylenediaminetetraacetate (EDTA); Fundamental and applied biological sciences. Psychology; Gene Expression; ligation-mediated PCR (LM-PCR); matrix-assisted laser-desorption ionization (MALDI) mass spectrometry; messenger RNA (mRNA); Molecular Sequence Data; nickel imino-diacetate agarose (Ni-IDA); phosphate-buffered saline (PBS); Phosphotransferases (Carboxyl Group Acceptor) - chemistry; Phosphotransferases (Carboxyl Group Acceptor) - genetics; Phosphotransferases (Carboxyl Group Acceptor) - metabolism; Phylogeny; polyacrylamide gel electrophoresis (PAGE); Polymerase Chain Reaction; polymerase chain reaction (PCR); Protozoa; recombinant carbamate kinase enzyme (TvCBK); Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; reverse-transcription PCR (RT-PCR); ribonuclease (RNase); RNA, Messenger - analysis; Sequence Analysis, DNA; sodium dodecyl sulfate (SDS); Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Systematics. Geographical distribution. Morphology. Cytology; Trichomonas vaginalis; Trichomonas vaginalis - classification; Trichomonas vaginalis - enzymology; Trichomonas vaginalis - genetics; Trichomonas vaginalis carbamate kinase gene sequence (TvCBK); ligation-mediated PCR (LM-PCR); nickel imino-diacetate agarose (Ni-IDA); recombinant carbamate kinase enzyme (TvCBK); matrix-assisted laser-desorption ionization (MALDI) mass spectrometry; Trichomonas vaginalis carbamate kinase gene sequence (TvCBK); phosphate-buffered saline (PBS); sodium dodecyl sulfate (SDS); carbamate kinase (CBK; ribonuclease (RNase); reverse-transcription PCR (RT-PCR); dithiothreitol (DTT); ethylenediaminetetraacetate (EDTA); EC 2.7.2.2; messenger RNA (mRNA); polyacrylamide gel electrophoresis (PAGE); polymerase chain reaction (PCR); carbamoyl phosphate synthetase (CPS); Trichomonas vaginalis; Protozoa; Host parasite relation; Protozoal disease; Nucleotide sequence; Enzyme; Taxonomy; Transferases; Interspecific comparison; Chemotaxonomy; Parasite; Parasitosis; Trichomonadida; Phylogeny; Gene expression; Infection; Trichomoniasis; Carbamate kinase; Recombinant protein; Aminoacid sequence
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1006/expr.2000.4507

Minotto, L., Edwards, M. R., and Bagnara, A. S. 2000. Trichomonas vaginalis: Characterization, expression, and phylogenetic analysis of a carbamate kinase gene sequence. Experimental Parasitology95, 54–62. 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 Km similar to that of the native enzyme and size-exclusion chromatography has shown that the enzyme is active as the homodimer.