Protein expression and purification
The SARS-CoV-2 receptor-binding domain (RBDWT) and the
RBD mutant (RBDK417N, RBDN501Y,
RBDK417N, RBDTRIP) were expressed using
Expi293F cells (Invitrogen, ThermoFisher Scientific, MA, USA). The genes
that encode SARS-CoV-2 RBDWT (residues Arg319-Phe541) or
RBD mutants with a C-terminal 6-His-tag was inserted into pTwist CMV
BetaGlobin WPRE Neo vector (Twist Bioscience, San Francisco, USA). The
construct plasmids were transfected into Expi293F cells using
ExpiFectatmine 293 transfection kit (Gibco, ThermoFisher Scientific, MA,
USA). The supernatant of cell culture containing the secreted RBDs was
purified using His-Trap HP column (GE Healthcare, USA). Collected
RBDWT or RBD mutated proteins were equilibrated in PBS
and kept at -20˚C. ACE2-mFc was purchased from Sino Biological
(Eschborn, Germany). Biotinylated and non-biotinylated soluble human
ACE2 fused to mouse IgG2a Fc proteins were kindly provided by PD Dr.
Alexander Eggel (University Clinic of Rheumatology and Immunology,
Inselspital, Bern, Switzerland) who received the plasmid from Prof.
Peter Kim (Stanford University, CA, USA).