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Obtaining and biochemical characterization of recombinant protozoan metacaspases of clinical interest


The present research project is a continuation of the previous projects carried out by our research group, in which we produced yeast metacaspases in an expression system in E. coli, in their wild-type forms and containing site-directed mutations. Interestingly, yeast metacaspases are classified as belonging to group I, which has the characteristic of presenting its N-terminal region constituted by apolar residues, contained a lot of residues of asparagines, glutamines and prolines. The biochemical characterization of these enzymes is compromised due to their insolubility, making it necessary to produce them without their N-terminal portion. Interestingly, protozoan metacaspases belongs to group II of these enzymes, which have the characteristic of not presenting the apolar portion at its N-terminal, being soluble and active in its monomeric form. In previous works by our group, we have already cloned, produced enzymes with site-directed mutagenesis and biochemically characterized the T.brucei metacaspase-2 (TbMCA-IIb). In this research project, we intend to obtain recombinant metacaspases from Trypanosoma cruzei, Leishmania amazonenses and Plasmodium falciparum, and with these recombinant enzymes we intend to evaluate their physicochemical characteristics, evaluating the interaction of calcium ions that are essential for the activation and activity of metacaspases, we carry out the complete biochemical characterization of these enzymes as an assay with synthetic substrates, study of pH and temperature. Such biochemical characterization will be important so that in future experiments with collaborators we can test and evaluate the action of molecules with the capacity to modulate the activity of mecaspases, being of great interest for the development of new drugs against these neglected diseases. (AU)

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