Cells were harvested by centrifugation at 3434 g for 20 min and resuspended in 20 mL of 50 mM Tris-HCl pH 8.0 and lysed via Emulsiflex (Avestin). To the extract was added NaCl, imidazole and β-mercaptoethanol for a final concentration of 250 mM, 10 mM and 10 mM, respectively. After lysis, cells were centrifuged at 11,000 g for 45 min at 4 °C to remove cell debris and the supernatant was loaded onto a nickel affinity resin (catalogue number Super-NiNTA25, Protein Ark), previously equilibrated with buffer [50 mM Tris-HCl pH 8.0, 250 mM NaCl, 10 mM imidazole and 5% (v/v) glycerol]. The purification was performed in a stepwise imidazole gradient and the purification fractions analyzed in an SDS-PAGE gel (Figure ). The fractions containing purified LCC-pExp-Bla recombinant protein were selected for concentration with Amicon Ultra-15 filters (Merck-Millipore, catalogue number UFC901024). To the concentrated material was added buffer containing 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2.5% (v/v) glycerol, and the material was submitted to another concentration step. This concentration and dilution step was repeated three times in order to remove the imidazole. To the final concentrated material was added 0.1 mg of TEV protease for cleavage overnight and the material was submitted to a centrifugation at 11,000 g for 15 min at 4 °C to remove any precipitated protein. The supernatant loaded into a column containing a resin previously equilibrated with buffer containing 100 mM NaCl, 100 mM bicarbonate pH 8 for a second purification step via IMAC.