Trichomonas vaginalis: expression and characterisation of recombinant S-adenosylhomocysteinase.
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).
Minotto
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School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, 2052, Australia.
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Edwards
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Bagnara
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D043383:Adenosylhomocysteinase; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006867:Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014246:Trichomonas vaginalis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D011994:Recombinant Proteins; D006867:Hydrolases; D043383:Adenosylhomocysteinase