The recombinant proteins expressed in E. coli BL21 (DE3) RIPL cells were purified according to previously described protocols (10, 12). Influenza HA4900 was purified from HEK293F suspension cells (Thermo Fisher Scientific, MA) according to previous reports (31). Briefly, the pAAV-ITRs-HA4900 plasmid was transiently transduced into HEK293F cells maintained in FreeStyle 293 expression medium (Thermo Fisher Scientific, MA) using the 293fectin Transfection Reagent (Thermo Fisher Scientific, MA). The cells were then incubated at 37°C and 8% CO2 while shaking at 130 rpm overnight. After 12 hours, an equal volume of fresh medium supplemented with sodium butyrate solution (enhancing protein expression; 2 nM final concentration) (Sigma-Aldrich, MO) was added to the cells. On day 7, the culture supernatants were harvested by centrifugation and filtered over 0.22-μm filters (Sartorius Stedim Biotech, Germany). Proteins were purified using HisTrap HP and gel filtration columns (GE Healthcare, IL).

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