Baculogold starter package kit

SF9 insect cells were grown in SF-900 II serum free media (Life Technologies). The cells were co-transfected with a pAcGP67 plasmid containing a H5 HA (A/Vietnam/1203/04 (H5N1), BEI Resources) expression construct and BD BaculoGold linearized baculovirus DNA (BD Biosciences). The H5 HA protein construct has an altered C-terminus, where the transmembrane and cytosolic regions of the protein were removed and replaced with an artificial trimerization domain (the foldon from T4 fibritin), and a His-tag as previously described (Stevens et al. 2006 (link)). Cell handling, transfection and protein expression were performed as recommended by the BD BaculoGold starter package kit (BD Biosciences). Viral titers were monitored using the BacPAK™ qPCR Titration Kit (Clontech Laboratories). For expression, fresh SF9 cells at 80 % confluency were infected with H5 HA containing baculovirus solution at MOI between 3 and 6. 4 days later the suspension was collected and the cells were removed by centrifugation. H5 HA is secreted into the insect cell media and purified by Ni-affinity chromatography. The protein concentrate was then subjected to Sephacryl S300 gel filtration column with phosphate buffer (50 mM sodium phosphate/pH 8.1 and 50 mM NaCl) as a running buffer. Protein fractions were pooled and concentrated and the final yield was ~4 mg of H5 HA per liter of insect cell culture.