Pgex 6p 1 expression vector

The 2B2t sequence (Hernández-González et al., 2012) [24 (link)] was subcloned into the pGEX-6P-1 expression vector (Cytiva, MA, USA) and a poly-His tag was added at the C-terminus. This construction was used to transform BL21-CodonPlus-RIL Escherichia coli competent cells (Agilent Technologies, Santa Clara, CA, USA). Protein expression was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma-Aldrich, Saint Louis, MO, USA) at 37°C and shaking (220 rpm) for three hours. A two-step affinity chromatography was performed for protein purification using two different sepharose resins. In the first step, GST-2B2t-poly-His recombinant protein was eluted from a Glutathione Sepharose 4B column (Cytiva, MA, USA) after incubation with L- Glutathione reduced (Sigma-Aldrich, St. Louis, MO, USA). Next, a second purification was performed on a Ni Sepharose 6 Fast Flow column (Cytiva, MA, USA) using 500 mM imidazole (Sigma-Aldrich, St. Louis, MO, USA) for elution. After dialysis against PBS, the purity, integrity and molecular weight of the protein were verified by SDS-PAGE (12%). The concentration was calculated with the Pierce BCA Protein Assay Kit (ThermoScientific, Pierce, IL, USA) and with a BSA protein standard curve (Fig 1). The recombinant protein was stored at -80°C until further usage.