We previously used a prefusion-stabilized, trimeric spike ectodomain (S2P ecto) to structurally define the sites several antibodies recognized on the SARS-CoV-2 spike trimer (Zost et al., 2020a (link)). This construct is similar to ones previously reported (Wrapp et al., 2020 (link)) and includes the ectodomain of SARS-CoV-2 (to residue 1,208), a T4 fibritin trimerization domain, and C-terminal 8x-His tag and TwinStrep tags. The construct also includes K986P and V987P substitutions to stabilize the spike in the prefusion conformation and a mutated furin cleavage site. S2P ecto protein was expressed in FreeStyle 293 cells (ThermoFisher) or Expi293 cells (ThermoFisher). Expressed S2P ecto protein was isolated by metal affinity chromatography on HisTrap Excel columns (GE Healthcare), followed by further purification on a StrepTrap HP column (GE Healthcare) and size-exclusion chromatography on TSKgel G4000SW XL (TOSOH). We also expressed a recently reported spike protein construct with 4 additional proline substitutions that enhance thermostability, yield, and structural homogeneity, here referred to as S6P ecto (Hsieh et al., 2020 ). The S6Pecto protein was expressed in FreeStyle293 cells and isolated on a StrepTrap HP column following the addition of BioLock Biotin Blocking Solution (IBA Lifesciences) to the culture supernatant.
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