TRICHOMONAS-VAGINALIS - EXPRESSION AND CHARACTERIZATION OF RECOMBINANT S-ADENOSYLHOMOCYSTEINASE

The gene encoding S-adenosylhomocysteinase activity (S-adenosylhomocys teine hydrolase, SAHH; EC 3.3.1.1) in Trichomonas vaginalis has been e xpressed in Escherichia coli to facilitate the characterisation of the enzyme. Expression of this gene using the pQE-30 (6xHis N-terminal ta g) expression system (QLAGEN) 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 enz yme has a molecular weight of approximately 56,000 and identification of tryptic peptides by matrix-assisted laser desorption ionisation (MA LDI) mass spectrometry has shown that the purified recombinant protein is identical in primary structure to the predicted sequence. The pres ence of the N-terminal 6xHis tag in the recombinant enzyme did not app ear to affect its kinetic and other properties, which are similar to t hose exhibited by the ''native'' enzyme present in cell-free extracts of T. vaginalis. These properties include a similar apparent K-m for a denosine (20-25 mu M for the recombinant and 5-10 mu M for the native enzymes, respectively) and similar inhibition/ inactivation patterns e xhibited by adenosine analogues such as arabinosyl adenine (ara-A). (C ) 1998 Academic Press.