Protein Expression and Purification.

Escherichia coli BL21(DE3) cells were transformed with a pET15b plasmid harboring the gene for BOA; the protein was expressed with an N-terminal hexa-His tag and purified as previously described (24). In short, 100 mL of Luria-Bertani (LB) broth containing 100 µg/mL of carbenicillin was inoculated with a single colony and incubated overnight at 37 °C. 10 mL of the overnight culture was added to 0.5 L of LB containing 100 µg/mL carbenicillin, followed by incubation at 37 °C until an optical density at 600 nm of 0.6 was achieved. Protein expression was then induced with 1 mM IPTG, followed by incubation at 18 °C for 16 h. Cells were harvested by centrifugation at 3,000 × g, and the cell pellet was resuspended in 50 mL of 20 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, 10 mM imidazole, and 1 mM TCEP. The cells were lysed using a microfluidizer, and cell debris was removed by centrifugation for 45 min at 18,000 × g. The supernatant was loaded onto a Cytiva HisTrap HP column and eluted using an imidazole gradient from 0 to 100% (20 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 500 mM imidazole, and 1 mM TCEP). Fractions containing BOA were dialyzed overnight at 4 °C against 50 mM Tris–HCL pH 8.0, 375 µM EDTA, and 1 mM TCEP, and the His-tag was cleaved by overnight incubation with tobacco etch virus protease (1:20 TEV to BOA ratio) and removed by passage over a HisTrap HP column. The flow-through containing BOA was concentrated to ~2 mL and loaded onto a S75 size-exclusion column and separated in 20 mM NaPO₄ pH 7.5. Fractions containing BOA were collected, concentrated, and frozen at −20 °C. The purity and identity of the BOA protein was assessed by SDS-PAGE and LC-MS.

E. coli BL21DE3 cells were transformed with a pET26b plasmid containing the gene for OAA, and the protein was expressed following the same procedure as was used for BOA. OAA was purified as previously described (22). Briefly, cells were pelleted at 3000 × g and resuspended in 20 mM Tris–HCl pH 8.5, lysed using a microfluidizer, and centrifuged at 18,000 × g for 45 min to remove cellular debris. This lysate was dialyzed overnight into 20 mM Tris-HCl pH 8.5 (at 25 °C) and loaded onto a Q HP anion exchange column and eluted using a 0-1 M gradient of NaCl + 20 mM Tris-HCl. Fractions were analyzed via SDS-PAGE and fractions containing OAA were concentrated using an Amicon spin concentrator. OAA was further purified by SEC chromatography using 20 mM Tris–HCl and 100 mM NaCl, pH 8.5 (at 25 °C). Fractions containing OAA were collected, concentrated, and frozen at −20 °C until further use.

The paH vector harboring the gene for Hexapro S was obtained from Addgene (Plasmid #166856) and used to transfect expi293F cells as previously described (30). Cells were induced for expression with 3.5 mM valproic acid, and media containing the secreted spike protein were collected after 3 d and clarified by centrifugation. The clarified supernatant was passed over a HisTrap HP column, and spike protein was eluted using a 0 to 100% gradient of elution buffer (20 mM NaH₂PO₄, pH 8.0, 150 mM NaCl, and 500 mM imidazole). Fractions were analyzed for the presence of spike protein on SDS-PAGE, and spike protein-containing fractions were pooled and concentrated to <1 mL. Further purification was carried out by size exclusion chromatography over a Superose-6 10/300 GL column in 20 mM NaPO₄, 150 mM NaCl, pH 7.5, and spike protein-containing fractions were pooled, concentrated, and flash frozen in 100 µL aliquots. The purity and identity of the spike protein was confirmed by SDS-PAGE. Negative stain electron microscopy was used to confirm the spike protein was in the prefusion conformation.