Histrap high performance ni sepharose columns

Each S₂ construct was produced in Expi293F cells (ThermoFisher Scientific) and cultured at 37°C in a humidified 8% CO₂ incubator with constant rotation at 130 RPM using Expi293 Expression Medium (ThermoFisher Scientific). DNA transfections were conducted using the ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) protocols and materials and cultivated for five days before harvest. Cell culture supernatants were clarified by centrifugation and proteins were harvested using HisTrap^™ High Performance Ni Sepharose columns (Cytiva). Proteins were washed using 10–15 CVs of buffer containing 25 mM Tris, 150 mM NaCl, 20 mM Imidazole pH 8.0 followed by elution with 10–15 CVs of buffer containing 25 mM Tris, 150 mM NaCl, 300 mM Imidazole pH 8.0. Eluates were buffer exchanged and concentrated into 20 mM Tris, 150 mM NaCl, pH 8.0 using Amicon Ultra-15 Centrifugal Filter Unit (10 kDa) (Millipore). Gel filtration was performed to remove unfolded or aggregated protein thus samples were each run through a Superose-6 Increase 10/300 GL column (Cytiva)) equilibrated in 20 mM Tris, 150 mM NaCl, pH 8.0. Main peaks were collected and protein was snap frozen and stored at −80 °C with some set aside for stability tests. Purified proteins for immunogenicity study were tested for endotoxin levels using Limulus Amebocyte Lysate (LAL) cartridges (Charles River PTS201F).