Expression and purification of recombinant FOXO3-DBD

Expression and Purification of FOXO3-DBD

DNA encoding human FOXO3-DBD (residues 156-269) was cloned into pGEX-6P-1 and expressed in E. coli BL21(DE3) as a fusion protein with the N-terminal GST affinity tag. Protein expression was induced using 0.5 mM β-D-1-thiogalactopyranoside and was done at 20°C for 16-20 hours. After centrifugation, cells (from 3 liters) were resuspended in 100 mL lysis buffer (20 mM phosphate at pH 7.4, 250 mM NaCl, 1 mM EDTA and 10 mM DTT) and stored in -80 °C. After thawing, cells were pretreated with lysozyme (100 µg/mL) for 1 hour and lysed by sonication at 4°C followed by centrifugation at 100,000 g for 45 min. Seven milliliters of Glutathione Sepharose® 4 Fast Flow (Merck, Austria), previously equilibrated in buffer 1 (20 mM Tris-HCl buffer, pH 7.5, 0.5 M NaCl, 1 mM EDTA and 10 mM DTT), was added to the supernatant, and the slurry was mixed for 1 hour at 4 °C. The resin was then poured into an Econo-Pac® Chromatography Column (Bio-Rad) and washed with 300 ml of buffer 1 using a peristaltic pump. Protein was eluted using 20 mL of 10 mM reduced glutathion in buffer containing 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 1 mM EDTA and 10 mM DTT followed by dialyses against buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA and 10% (wt/vol) glycerol. The fusion protein was cleaved during the dialysis using PreScission protease (10 U/mg of fusion protein) at 4° overnight. After cleavage, protein solution was concentrated using Vivaspin® Turbo 15 ultrafiltration unit and subjected to size-exclusion chromatography on a HiLoad Superdex 75 26/600 column (GE Healthcare) with 20 mM phosphate buffer (pH 6.5) containing 1 mM DTT, 50 mM KCl and 10% (wt/vol) glycerol as a mobile phase. Fractions containing FOXO3-DBD were collected and concentrated to 5-7 mg/mL. The purified protein was characterized by SDS-PAGE.