The stable cell line generation vector pNeoSec was used for cloning of the SARS-Cov2 Spike ectodomain comprising amino acids 27-1208 with mutations of the furin cleavage site (RRAR > GSAS at residues 682-685) and the PP (KV > PP at residues 986-987). At the N terminus, there is a twin StrepII tag and at the C terminus fused with a T4 fibritin trimerisation domain, an HRV 3C cleavage site and a His-8 tag. The human embryonic kidney (HEK) Expi293F cells (Thermo Fisher Scientific) were transfected with the construct together with a phiC31 integrase expression plasmid as described earlier (Zhao et al., 2014). The polyclonal G418 resistant (1 mg/ml) cell population were used for protein production. Expi293F cells were grown in adhesion in roller bottles with the high glucose DMEM (Sigma) with 2% FBS for 6 days at 30°C. The soluble spike protein was captured from the dialysed conditional media with prepacked 5 mL Columns of HisTrap excel (GE Healthcare Life Sciences). The protein was eluted in 300 mM imidazole containing phosphate-buffered saline (PBS) after a 20 mM imidazole PBS wishing step. The protein was further purified with a 16/600 Superdex 200 size exclusion chromatography with an acidic buffer (20 mM Acetate, 150 mM NaCl, pH 4.6) for the low pH Spike incubations, or a neutral buffer (2 mM Tris, 150 mM NaCl, pH 7.5).

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