**2.2 Purification of recombinant TcISP2 (TcISP2r) from** *Escherichia coli*

The construction of recombinant plasmid to express TcISP2r in *E. coli* and extraction of TcISP2r were previously described by our research group [44]. Initially, a culture of *E. coli* from DH5α strain was transformed with pET28a plasmid containing TcISP2r construction with a histidine tag at N-terminus, in the presence of 50 ug/ mL of Kanamycin at 600 nm optical density of 0,5, following expression induction with 1 mM of IPTG and incubation for more 16 hours in the shaker at 37<sup>⁰</sup> C. For protein extraction, 50 mL of bacteria culture was precipitated by centrifugation and the pellet was dissolved in 6 mL of 20 mM of phosphate buffer pH 7.4 and 0.024 g of lysozyme (Sigma, USA), and incubated at room temperature for 15 minutes. Then 6 ml of lysis buffer (20 mM phosphate buffer pH 7.4 and 0.5% Tween 20) was added and incubated for 30 minutes at 37°C. Finally, the bacterial lysate was centrifuged at 8000 x g for 15 minutes at 4°C to precipitate cell debris. The protein present in the supernatant was purified on an affinity chromatography column containing nickel, HisPur Ni-NTA Spin 0.2 ml (Thermo Fisher Scientific, USA), following the supplier's instructions. After passing the entire supernatant through the column by successive centrifugations at low speed (4000 x g) for 1 min, the column was washed with 10

*Exploring the Evolutionary Origin and Biological Role of the* Trypanosoma cruzi *Ecotin-Like… DOI: http://dx.doi.org/10.5772/intechopen.109929*

volumes of 20 mM phosphate buffer pH 7.4 with 20 mM Imidazole. Subsequently, the protein was eluted from the column with 150 mM Imidazole. The purified TcISP2r protein was subjected to concentration by filtration on 10 KDa cellulose filters to replace the 20 mM phosphate buffer pH 7.4 containing 150 mM imidazole with 20 mM phosphate buffer pH 7.4 without imidazole. The final protein concentration was determined by absorbance at 280 nm in a UV light spectrophotometer. The quality and quantity of protein obtained were also verified by protein electrophoresis in 12% acrylamide gel. Protein specificity was verified by Western Blotting using an anti-histidine antibody.
