Cloning and expression of recombinant L19 ribosomal protein from Trypanosoma cruzi.

JUAN M ZÁRATE; WALTER J. LAPADULA; NATALIA CARMONA; MAXIMILIANO JURI AYUB

Congreso; XXVIII Reunión de la sociedad de biología de Cuyo; 2010

Trypanosomatids are a monophyletic group of protozoa that diverged very early during evolution, comparing to model eukaryotic organisms such as plants, yeast and mammals. Trypanosoma cruzi acquires particular importance in Latin America as the etiologic agent of Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. There are important evidences suggesting that the translational system in trypanosomatids shows important differences respect other eukaryotes, in particular mammals. Previously, using data mining and mass spectrometry, we have shown that T. cruzi ribosomal protein L19 (TcL19) has a C-terminal extension of 168 amino acids, with no similarity to any known domain, and absent from organisms other than trypanosomatids. Here, we report the cloning and expression of recombinant TcL19 in E. coli, in two different versions; full-length and without the T. cruzi-specific C-terminal domain. Both proteins were cloned and expressed correctly. Interestingly, the full-length, but not the truncated version, was toxic for E. coli cells. Currently, we are setting the purification protocol for the truncated protein, in order to obtain specific anti-sera for detecting native TcL19 in T. cruzi epimastigotes by Western Blot and indirect inmunifluorescence (IFI). This will allow to known whether TcL19 is processed before the ribosome assembly or the C-terminal domain is present in mature ribosomes.