All wild-type recombinant (HA) constructs were expressed by infection of insect cells with recombinant baculovirus as previously described. In brief, synthetic DNA corresponding to the full-length ectodomain were subcloned into a pFastBac vector modified to encode a C-terminal thrombin cleavage site, a T4 fibritin (foldon) trimerization tag, and a 6×His tag (19, 39). The resulting baculoviruses produce HA-trimers. Supernatant from recombinant baculovirus infected High Five Cells (Trichoplusia ni) was harvested 72 h postinfection and clarified by centrifugation. Proteins were purified by adsorption to cobalt-nitrilotriacetic acid (Co-NTA) agarose resin (Clontech), followed by a wash in 10 mM Tris⋅HCl, 150 mM NaCl at pH 7.5 (buffer A), elution in buffer A plus 350 mM imidazole (pH 8) and gel filtration chromatography on a Superdex 200 column (GE Healthcare) in buffer A.

HA proteins for immunization were prepared in buffers made with endotoxin-free water (HyClone HyPure Cell Culture Grade Water; SH30539.03). Following gel filtration, the tags were removed using thrombin protease (Thrombin CleanCleave Kit; Sigma; catalog #RECOMT-1KT) and the protein repurified on Co-NTA agarose to remove the protease, tag, and uncleaved protein. The protein was concentrated and buffer exchanged into Endotoxin-Free Dulbecco’s PBS (EMD Milipore; TMS-012-A) and further purified using gel filtration chromatography on Superdex 200 (GE Healthcare) in PBS to further remove impurities. Endotoxins were removed using Pierce High Capacity Endotoxin Removal Spin Columns (catalog #88274). Proteins were then concentrated and sterile filtered.

All mutant HAs were produced from synthetic DNAs that corresponded to the to the full-length ectodomain (FLsE) or the globular HA-head. These were cloned into a pVRC vector that has been modified to encode a C-terminal thrombin cleavage site, a T4 fibritin (foldon) trimerization tag, and a 6×His tag. The resulting proteins are rHA trimers and trimeric HA heads. All were produced by polyethylenimine-facilitated, transient transfection of 293F cells that were maintained in FreeStyle 293 Expression Medium. Transfection complexes were prepared in Opti-MEM and added to cells. Supernatants were harvested 4–5 d posttransfection and clarified by low-speed centrifugation. Subsequent purification was then identical to those for wild-type insect cell-produced HAs.

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