SARS-CoV-2 Spike Protein Production

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 (link)). The S6P_ecto 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.