Improvement in the production of active cruzain for in vitro inhibitors screening assays

Abstract

Chagas disease is a chronic systemic parasitic infection caused by the protozoan parasite Trypanosoma cruzi. A promising target for potential therapeutics against it is the major cysteine protease expressed by T. cruzi, cruzain. Cruzain is a member of the papain superfamily of cysteine proteases, and it is homologous to cathepsin-L. These enzymes degrade polypeptides and are characterized by having a common catalytic mechanism that involves a nucleophilic cysteine thiol in the catalytic triad. In the case of Chagas disease, cruzain is present in all life stages of the trypanosome and is implicated in cellular entry and digestion of immunoglobins. Inhibition studies of cruzain in cell models indicated prevention and reduction of infection.Previously, we demonstrated the expression of a recombinant cruzain in E. coliBL21(DE3) classic, pLys E, and Rosetta strains.1 However, most of the produced protein was present as protein aggregates. Optimization of culture conditions, including inductor concentrations and temperature shifts, allowed us to maximize the production of soluble active cruzain in concentration, yield and purity suitable for in vitro high throughput screening assays to identify potentially new and novel cruzain inhibitors by both kinetic and endpoint assays. Preliminary fluorometric assays showed that the mature cruzain cleaves the synthetic fluorogenic peptide substrate N-CBZ-Phe-Arg-7-amido-4-methylcoumarin under our experimental conditions. With the aim of identifying new chemical scaffolds leading to the development of novel drugs useful in the treatment ofChagas' disease, we offer this assay for joint research activities and collaborations.