Cobalt affinity column

The SARS-CoV S^B construct was cloned from a SARS-CoV S ectodomain (Walls et al., 2019 (link)) synthesized by GeneArt (ThermoFisher Scientific) into a modified pOPING vector with an N-terminal mu-phosphatase signal peptide and a C-terminal hexa-histidine tag (G-HHHHHH). The boundaries of the construct are N-terminal ₃₀₆RVVPSG₃₁₁ and C-terminal ₅₇₁LDISP₅₉₇₅. The SARS-CoV-2 S^B construct was synthesized by GenScript into pcDNA3.1-with an N-terminal mu-phosphatase signal peptide and a C-terminal hexa-histidine tag (G-HHHHHH). The boundaries of the construct are N-terminal 328RFPN₃₃₁ and C-terminal 530STNL₅₃₃. Both constructs were produced in 500mL HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life technologies) at 37°C in a humidified 8% CO₂ incubator rotating at 130 rpm. The cultures were transfected using 293-Free transfection reagent (Millipore) with cells grown to a density of 1 million cells per mL and cultivated for 3–4 days. The supernatants were harvested and cells resuspended for another 3–4 days, yielding two harvests. Proteins were purified from clarified supernatants using a 5mL Cobalt affinity column (Takara), concentrated and flash frozen in a buffer containing 20 mM Tris pH 8.0 and 300 mM NaCl prior to analysis. SDS-PAGE was run to check purity.

All ectodomains were produced in 500mL cultures of HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life technologies) at 37°C in a humidified 8% CO2 incubator rotating at 130 r.p.m., as previously reported^{18 (link)}. The culture was transfected using 293fectin (ThermoFisher Scientific) with cells grown to a density of 10⁶ cells per mL and cultivated for three days. The supernatant was harvested and cells were resuspended for another three days, yielding two harvests. Clarified supernatants were purified using a 5 mL Cobalt affinity column (Takara). Purified protein was concentrated, and flash frozen in a buffer containing 50 mM Tris pH 8.0 and 150 mM NaCl prior to cryo-EM analysis.