Purification and Characterization of SARS-CoV-2 Spike Glycoprotein

The S glycoprotein ectodomain was expressed and purified using a previously described protocol^{17 (link)}. The construct was transformed into bacterial DH10Bac competent cells (Invitrogen); then, the extracted Bacmid was transfected into Sf9 cells (source: American Type Culture Collection) using Cellfectin II Reagent (Invitrogen). The passage 1 (P1) baculoviruses were harvested and amplified to generate a high-titer virus stock with Sf9 cells, and then they were used to produce the recombinant proteins. The supernatant of the cell culture containing the secreted S glycoprotein was harvested at 60 h after infection and concentrated, and the buffer was changed to binding buffer (10 mM HEPES, pH 7.2, 500 mM NaCl). Finally, S glycoprotein was captured by StrepTactin Sepharose High Performance (GE Healthcare) and eluted with 10 mM D-desthiobiotin in binding buffer. Oligomerization of the HCoV-229E S trimer (1 mg) was analyzed using a Superose 6 Increase 10/300 GL column (GE Healthcare) with a buffer containing 10 mM HEPES, pH 7.2, and 150 mM NaCl^{23 (link)} at a flow rate of 0.3 ml/min (4 °C). We found three peaks in the gel filtration profile (Supplementary Fig. 1a). Because peak 1 represents a highly aggregated state, we collected the fractions of peaks 2 and 3 for the cryo-EM analysis. The proteins collected in the different fractions were analyzed by SDS-PAGE.