Stericup gp device

The aerial parts of the plants were harvested 5 days post agroinfiltration and homogenized in two buffer volumes of PBS (Lonza), supplemented with cOmplete^™ EDTA-free protease inhibitor (Roche). The crude homogenate was incubated for 1 h, at 4°C, with shaking and then filtered through four layers of Miracloth^™ (Merck). The crude plant sap was then clarified by sequential centrifugation steps; twice at 15,344 × g for 20 min and then again at 17,000 × g for 20 min. The supernatant was vacuum-filtered through a 0.45 µM Stericup-GP device (Merck Millipore) and applied to a Galanthuis nivalis lectin (GNL) column (Sigma) with a 0.5–1 ml/min flow rate. The column was sequentially washed with 100 ml of 0.5 M NaCl and then 100 ml of PBS (Lonza). The proteins were eluted in 1 M methyl α-D-manno-pyranoside (MMP) (Sigma), buffer exchanged into PBS and then concentrated using a Vivaspin Protein Concentrator with a 30 kDa cut-off (GE Healthcare). The purified proteins were quantified using the DC^™ Protein Assay (Bio-Rad).

FreeStyle™ HEK293F cells (Invitrogen) were grown in sterile polycarbonate Erlenmeyer flasks on an orbital shaking platform set to 125 rpm. The cells were maintained at a density of 1-3×10⁶ cells/ml at 37°C, with 8% CO₂. The cultures were passaged every 3-4 days, at a seeding density of 3×10⁵ cells/ml, using fresh FreeStyle™ 293 Expression Medium (Invitrogen). Spike protein was transiently expressed by transfecting cells, at a density of 1×10⁶ cells/ml, with 1 µg/ml of plasmid DNA. Polyethylenimine was used for transfections at a 3:1 ratio of transfection reagent:DNA. The culture media was harvested 5 days post-transfection and clarified by centrifugation at 2500×g, for 30 minutes. The clarified media was then filtered using a 0.45 µm Stericup-GP device (Merck Millipore). Spike trimers were purified as described for the plant-produced material.