MEDIUM-SCALE PRODUCTION AND PURIFICATION TO HOMOGENEITY OF A RECOMBINANT TRANS-SIALIDASE FROM TRYPANOSOMA-CRUZI

Trypanosoma cruzi, the agent of Chagas' disease, presents an enzyme th at catalyzes the transfer of sialic acid among glycoproteins and glyco lipids known as trans-sialidase (TS), displaying some interesting feat ures: 1) It differs from all other eucaryotic sialyltransferases, both kinetically and in substrate specificity and 2) it is involved in the parasite's mechanism of mammalian host cell invasion. We report here the production and purification to homogeneity of an enzymatically act ive recombinant TS (rTS) lacking the C-terminal amino acid repeats, us ing iminodiacetic metal affinity chromatography. Initial ratios of non -fusion recombinant versus total protein were very low in several expr ession systems tested, mainly due to high degradation rate. However, a fter purifying 1,330 times, we were able to obtain an essentially homo geneous preparation of rTS with a final yield of 29%. After minor chan ges, a modified protocol for a medium scale production was designed ob taining 0.5 mg of homogeneous rTS per liter of bacterial culture. The purified rTS behaved as a homogeneous protein in silver-stained denatu ring gels, isoelectrofocusing and N-terminal sequencing having identic al pH and temperature optima as the natural enzyme. Conditions to keep the rTS for long periods without a significant loss of activity were identified.