Cloning of Trypanosoma cruzi trans-Sialidase and Expression in Pichia pastoris Wouter Laroy and Roland Contreras 1 Department of Molecular Biology, Unit of Fundamental and Applied Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium Received April 3, 2000, and in revised form August 9, 2000 Trypanosoma cruzi, the agent causing Chagas’ dis- ease, 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 eukary- otic sialyltransferases in that it does not accept cyti- dine 5-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltrans- ferase 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 pas- toris. 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 ob- tained about 5 mg of enzymatically active trans-siali- dase per liter of induced culture medium. © 2000 Academic Press Key Words: Trypanosoma cruzi; trans-sialidase; het- erologous expression; Pichia pastoris. Trypanosoma cruzi is the parasite that causes Amer- ican trypanosomiasis or Chagas’ disease. The organism is most commonly found in Central and South America, where it affects approximately 18 million people. It is a digenic organism with life stages in both insect vectors and mammals (1). Although these parasites are not able to synthesize sialic acid themselves, the sugar residue was identified on their cell surfaces (2, 3). An enzyme called trans-sialidase was found to be respon- sible for this activity. It was first characterized and cloned in T. cruzi (4, 5), and later in different Trypano- soma brucei subspecies (6) and Trypanosoma congo- lense (7). Except for the highly related flagellate En- dotrypanum (8), no other organisms were found to express such enzyme. Although the presence of the enzyme is essential for the pathogenicity of the Trypanosoma species, the ex- act function was not revealed yet. Evidence was pre- sented to suggest a role in cell invasion and attachment to the host cell (9). However, for this event, only siali- dase activity seems to be of importance (10). More probably, trans-sialidase is involved in the escape from host protection mechanisms (11). Chagastic anti--Gal immunoglobulins expressed by the host are responsi- ble for both agglutination and lysis of Trypanosoma cells (10). Trans-sialidase was also suggested to be a virulence factor (12). Different genes encoding T. cruzi trans-sialidase were cloned previously (13–15). They belong to a family of about 80 copies per haploid genome, organized in clusters of up to 12 repeats (16). The catalytic domain is located N-terminally. Apart from the catalytic site, this domain contains different Asp box sequences, which are motifs commonly found in prokaryotic and eukaryotic sialidases. A C-terminal repeat region can be present, but is not essential for enzymatic activity. When present, it is involved in oligomerization (17) and improves the stability of the protein (18). Also, this domain carries the motif needed for the attachment of a GPI 2 anchor. The structure of a trans-sialidase ho- molog from Trypanosoma rangeli was solved recently (19). As expected, a typical sialidase fold was found. However, this enzyme only has sialidase activity. Based on this structure, a model was proposed for trans-sialidase, but evidence to support this model is lacking. To study this enzyme, high amounts of easily obtain- able trans-sialidase are needed. Until now, the protein 1 To whom correspondence should be addressed. Fax: (32 9) 264 87 98. E-mail: roland.contreras@dmb.rug.ac.be. 2 Abbreviations used: AOX1, alcohol oxidase; bp, basepairs; DEAE, diethylaminoethyl; dNTP, deoxy nucleotide triphosphate; Gal, galac- tose; GPI, glycosyl phosphatidyl inositol; PCR, polymerase chain reaction. Protein Expression and Purification 20, 389 –393 (2000) doi:10.1006/prep.2000.1334, available online at http://www.idealibrary.com on 389 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.