Before the BLI experiments, SARS-CoV-2 S trimer protein was biotinylated using the EZ-Link Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher Scientific) and then purified using the Zeba spin desalting column (Thermo Fisher Scientific) according to manufacturer’s protocols. To determine binding affinity of ACE2, BLI assay was carried out using an Octet Red 96 instrument (Pall FortéBio, USA). Briefly, biotinylated SARS-CoV-2 S trimer protein was loaded onto streptavidin biosensors (Pall FortéBio). S-trimer–bound biosensors were dipped into wells containing varying concentrations of ACE2 protein, and the interactions were monitored over a 500-s association period. Finally, the sensors were switched to dissociation buffer [0.01 M phosphate-buffered saline (PBS) supplemented with 0.02% Tween 20 and 0.1% bovine serum albumin] for a 500-s dissociation phase. Data were analyzed using Octet data analysis software version 11.0 (Pall FortéBio).

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