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Fig. 3. Overexpression of functional chagasin in transfected T. cruzi. (A) Epimastigotes were washed twice in PBS and lysed in PBS containing 1% Triton X-100. The lysates (100 µg) were resolved by SDS-PAGE and submitted to western blot using anti-chagasin antiserum (1:1000). Anti-calreticulin monoclonal antibodies were used as a control for sample loading in the gels. (B) Inhibitory activity in transfected epimastigotes. Lysates (2.4 µg) were boiled for 20 minutes and the soluble fraction was collected after 10,000 g centrifugation. Equal volumes of the soluble fraction were incubated with papain (3.32 nM) in 50 mM Na₂PO₄, 100 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT for 15 minutes at room temperature and the residual activity was subsequently monitored by the hydrolysis of 5 µM CBZ-Phe-Arg-AMC in the same buffer containing 5% DMSO. As a control, papain was incubated for the same period in buffer and the peptidase activity was measured in the same conditions. (C) Chagasin overexpression leads to a reduction in CP activity. The peptidase activity contained in normalized epimastigote lysates (1 µg) was determined in 50 mM Na₂HPO₄, 200 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT, 5% DMSO at room temperature, using 5 µM CBZ-Phe-Arg-AMC as a substrate. Substrate hydrolysis was fully inhibited by 10 µM E-64 (data not shown), confirming that the activity detected corresponds to that of CPs. The graph shows the initial velocities for the substrate hydrolysis curves. The experiments were performed in triplicate and are represented as mean values with standard deviations (SD). The analysis of variance was performed using ANOVA and the asterisk indicates the scores that are statistically significant at P<0.05 (B) or P<0.001 (C).

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