Trichomonas vaginalis:Expression and Characterisation of RecombinantS-Adenosylhomocysteinase

Abstract

Minotto, L., Ko, G.-A., Edwards, M. R., and Bagnara, A. S. 1998.Trichomonas vaginalis: Expression and characterisation of recombinantS-adenosylhomocysteinase.Experimental Parasitology90, 175–180. The gene encodingS-adenosylhomocysteinase activity (S-adenosylhomocysteine hydrolase, SAHH; EC 3.3.1.1) inTrichomonas vaginalishas been expressed inEscherichia colito 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 ofT. vaginalis.These properties include a similar apparentKmfor adenosine (20–25 μM for the recombinant and 5–10 μM for the native enzymes, respectively) and similar inhibition/inactivation patterns exhibited by adenosine analogues such as arabinosyl adenine (ara-A).