S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen

Makadiya, Niraj; Brownlie, Robert; van den Hurk, Jan; Berube, Nathalie; Allan, Brenda; Gerdts, Volker; Zakhartchouk, Alexander

Purifications of supernatants from the recombinant yeast cells and recombinant baculovirus infected cells were performed in the same way. First, equal volume of wash buffer was added to the samples (50 mM sodium phosphate, 0.3 M sodium chloride, 10 mM imidazole pH 8.0) and then pH was adjusted to 8.0. Next, the samples were passed through 0.2 μm filters to remove cellular debris and applyed to the HisSelect Ni Affinity (Sigma Aldrich) column. The column was washed with three sample volumes of wash buffer, followed by protein elution in one sample volume of elution buffer (50 mM sodium phosphate, 0.3 M sodium chloride, 250 mM imidazole pH 8.0). The eluate was concentrated using the Amicon Ultra-15 (EMD Millipore) filters and protein concentration was determined by a Bradford assay, and the protein quality was analysed by Western blotting.