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 that catalyzes the transfer of sialic acid among glycoproteins and glycolipids known as trans-sialidase (TS), displaying some interesting features: 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 active recombinant TS (rTS) lacking the C-terminal amino acid repeats, using iminodiacetic metal affinity chromatography. Initial ratios of non-fusion recombinant versus total protein were very low in several expression systems tested, mainly due to high degradation rate. However, after purifying 1,330 times, we were able to obtain an essentially homogeneous preparation of rTS with a final yield of 29%. After minor changes, a modified protocol for a medium scale production was designed obtaining 0.5 mg of homogeneous rTS per liter of bacterial culture. The purified rTS behaved as a homogeneous protein in silver-stained denaturing gels, isoelectrofocusing and N-terminal sequencing having identical pH and temperature optima as the natural enzyme. Conditions to keep the rTS for long periods without a significant loss of activity were identified.
Buschiazzo
A
A
Instituto de Investigaciones Bioquímicas, Fundación Compomar, Buenos Aires, Argentina.
Frasch
A C
AC
Campetella
O
O
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D009439:Neuraminidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D006023:Glycoproteins; D011994:Recombinant Proteins; C069715:trans-sialidase; D009439:Neuraminidase