Cloning of Trypanosoma cruzi trans-sialidase and Expression in Pichia pastoris

Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme t hat transfers sialic acids among glycoproteins and glycolipids both from th e host cell surface and its own surface. This enzyme, called trans-sialidas e, is different from higher eukaryotic sialyltransferases in that it does n ot accept cytidine 5'-monophospho-N-acetylneuraminic acid as a donor substr ate. Also, the common glycosyltransferase structure is not present. To stud y this enzyme, an active member was cloned and expressed in higher eukaryot ic cells. Expression of recombinant enzyme was achieved in the methylotroph ic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and t he C-terminal addition of an epitope tag resulted not only in high expressi on levels, but also enabled easy detection and purification. Using P. pasto ris, we obtained about 5 mg of enzymatically active trans-sialidase per lit er of induced culture medium, (C) 2000 Academic Press.