Scalable Production of Recombinant Ectodomains

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.