Cloning of Trypanosoma cruzi trans-sialidase and expression in Pichia pastoris.
Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface. This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5'-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltransferase structure is not present. To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells. Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification. Using P. pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium.
Laroy
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Department of Molecular Biology, Unit of Fundamental and Applied Molecular Biology, Ghent University, B-9000 Ghent, Belgium.
Contreras
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D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular / Q000379:methods*; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D009439:Neuraminidase / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014349:Trypanosoma cruzi / Q000235:genetics*
D006023:Glycoproteins; D011994:Recombinant Proteins; C069715:trans-sialidase; D009439:Neuraminidase