Expression and purification of H protein

Pre-confluent (30–40%) monolayers of HEK293T cells were transfected with pDisplay-H-ICV using a standard calcium phosphate transfection protocol described elsewhere. Following transfection, cells were cultured at 37°C for up to 8 days and supernatants were collected every 48 h. Supernatants were clarified by ultracentrifugation for 30 min at 3,000 x g, filtered through 0.45 µm filter (Millipore) and subsequently loaded onto an anti-HA sepharose coupled column at 0.5 ml/min flow rate. Column was washed with 5 column volumes of 10 mM Tris-HCl pH 8.3, 100 mM NaCl buffer to remove unspecific bound proteins. Protein was eluted with 20 mM Glycine pH 3 in 7 ml fractions. Fractions were analyzed by SDS-PAGE and the fraction showing the highest concentration was further analyzed by size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare) in the same buffer.