Characterization and expression of a putative enzyme of the thiol metabolism in Trypanosoma cruzi

AINCIART, N; GARCIA, GA; POTENZA, M; RUIZ, AM

Villa Carlos Paz, Cordoba, Argentina

Congreso; XXXVIII Reunion Nacional de la Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular; 2002

Sociedad Argentina de Investigacion Bioquimica

Thiol metabolism divergences between Trypanosoma cruzi, Chagas’ disease ethiological agent, and its mammalian host lead to postulate this pathway’s enzymes as potential targets for tripanocidal drugs. We identified the TcGRX gene, which codifies a protein homologous to glutaredoxins and glutathione-S-tranferases, with no homology to mammal enzymes. TcGRX sequence homologies and the structure obtained by molecular modeling onto E. coli Grx 3 suggest it might be implicated in thiol metabolism. In order to obtain recombinant TcGRX, this gene was cloned in pRSET (N-terminal His-tag), pET 28 and pET 43 (both with a C-terminal His-tag) and expressed in four different E. coli strains. Induction of soluble protein was evaluated by western blot with an anti-His antibody. Soluble TcGRX from pRSET was induced only in BL21(DE3)pLys and purification could only be achieved under denaturing conditions suggesting the N-terminal His-tag might be hidden. TcGRX from pET 28 was always insoluble. Soluble fusion protein NUS-TcGRX expressed by pET 43 was obtained in BL21(DE3)pLys and ORIGAMI, and could be purified by IMAC in native conditions. Difficulties observed through TcGRX expression demonstrate that solubility of recombinant proteins are highly dependent on the plasmid-host system used and encourage the idea to follow a multicloning strategy to reach success. Biochemical characterization of NUS-TcGRX might let us design specific inhibitors to evaluate their tripanocidal activity.