Purification of SARS-CoV-2 Spike Proteins and Antibodies

Proteins were expressed in Expi293 cells by transient transfection. IgGs and a previously described human ACE2-Fc construct¹¹ (link) were purified from cell supernatants using MabSelect SURE columns (Cytiva), and His-tagged Fabs were isolated from cell supernatants using Ni-NTA columns (Qiagen). IgGs, ACE2-Fc, and Fabs were further purified by SEC using a HiLoad 16/600 Superdex 200 column (Cytiva). Purified proteins were concentrated using a 100 kDa and 30 kDa cutoff concentrator (EMD Millipore), respectively, to 10 to 15 mg/mL, and final concentrated proteins were stored at 4 °C until use. 6P versions²⁵ (link) of soluble SARS-CoV-2 WA1 and SARS-CoV-2 Omicron BA.1 spike trimers were isolated from cell supernatants using a pre-packed Ni-NTA column (Cytiva). Eluents from Ni-NTA purifications were subjected to SEC using a HiLoad Superdex 200 16/600 column followed by a Superose 6 10/300 (Cytiva) column. Peak fractions were pooled and concentrated to ∼6 mg/ml, flash frozen in 50 μL aliquots, and stored at -80 °C until use.