TcPI3K: A class I PI-3 kinase in Trypanosoma cruzi.

GIMENEZ AM; SCHOIJET AC; TORRES HN; FLAWIÁ MM; ALONSO GD; MACHADO EE

Santa Fe, Argentina

Congreso; XXIII Reunión Científica Anual Sociedad Argentina de Protozoología.; 2009

Phosphatidylinositol kinases play a central role in different processes such as membrane trafficking, autophagy and cell signaling. Previously, we found that Trypanosoma cruzi epimastigotes possesses a PI3K activity which is a component in the signaling pathways activated by hyperosmotic stress. However, we did not identify the class of PI3K involved in this response. The aim of this work was to define if class I PI3K is functional in this parasite, through cloning and expressing a putative T. cruzi class I PI3K (TcPI3K) in BL 21 E. coli cells. Primer sequences were designed from the identified sequence (Tc00.1047053510167.10) and PCR product was sequenced and compared with the original sequence found at GeneDB. We used pET22 b(+) expression vector and protein induction with IPTG was performed at 25ºC to obtain enough soluble protein for its purification. Southern and Northern blot analysis showed that TcPI3K is a single copy gene and its mRNA is expressed in epimastigote forms. Moreover, recombinant TcPI3K recovered from soluble fraction was assayed in order to determinate kinase activity, confirming a functional PI3K. This is the first report of class I PI3K existence in trypanosomas.