EXPRESSION, PURIFICATION AND PRELIMINARY STRUCTURAL STUDIES OF CRUZAIN PROTEIN.

CARMONA N; ENRIZ D; GÓMEZ BARROSO JA; BALDONI HA; AGUILAR CF

Encuentro; XXXIV Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2016

Sociedad de Biología de Cuyo

Chagas? disease is a chronic systemic parasitic infection caused by the protozoan parasite Trypanosoma cruzi. Available drugs are highlytoxic and often ineffective, particularly those used to treat the chronic stage of the disease. The aim of our work is the structural andfunctional study of proteins which are vital for Trypanosoma cruzi as a first step in rational drug design. Cruzain protein is a member ofthe papain/cathepsin-L family of cysteine proteases, and the major cysteine protease of the protozoan T. Cruzi. The protein is present inall life stages of the trypanosome and is implicated in cellular entry and digestion of immunoglobins. . E. coli BL21 (DE3) Star weretransformed with pET21a-cruzain vector and single colonies were selected in LB agar containing specific antibiotic. A starter culture ofBL21(DE3) Star- TcCruzain was grown in 5 ml LB medium containing 50 mg/ ml ampicillin al 37 °C overnight. This initial culture wasincreased to 1 l, induced with IPTG at a concentration of 1 mM and grown for an additional 72h at 20°C. The bacteria were recovered bycentrifugation. His-TcCruzain was purified using Ni-Sepharose affinity column. Autoproteolysis of the pro-region (~ 14 kDa) from thezymogen (~ 37 kDa) was initiated by adding DTT (1 mM final concentration) to the solution and incubating in a 37 °C water bath.Transformation of the inactive zymogen into the catalytically active domain was monitored by removing 50 uL aliquots of the solution atselected time points. All stages have been analyzed by SDS-PAGE and Western Blot. The preliminary results show a high concentrationof active enzyme with a high degree of purity which would allow structural studies as the next step.