Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2

https://doi.org/10.1016/j.bbrc.2020.11.055Get rights and content

Highlights

  • 1.A high-throughput, 384-well plate assay was developed to measure the binding S1 RBD to ACE2;

  • 2.HiBIT-RBD binds to cells expressing ACE2 specifically and in a time dependant fashion;

  • 3.The binding of HiBIT-RBD to ACE2 can be inhibited using recombinantly expressed SARS-CoV-2 RBD and full-length, Spike S1;

  • 4.Site specific mutations within the RBD demonstrate the specificity of this assay.

Abstract

To identify drugs that could potentially be used to treat infection with SARS-CoV-2, a high throughput 384-well assay was developed to measure the binding of the receptor binding domain (RBD) of the viral S1 protein to its main receptor, angiotensin converting enzyme 2 (ACE2). The RBD was fused to both a HiBIT tag and an IL6 secretion signal to enable facile collection from the cell culture media. The addition of culture media containing this protein, termed HiBIT-RBD, to cells expressing ACE2 led to binding that was specific to ACE2 and both time and concentration dependant, Binding could be inhibited by both RBD expressed in E. coli and by a full length S1 - Fc fusion protein (Fc-fused S1) expressed in eukaryotic cells. The mutation of residues that are known to play a role in the interaction of RBD with ACE2 also reduced binding. This assay may be used to identify drugs which inhibit the viral uptake into cells mediated by binding to ACE2.

Keywords

SARS-CoV-2
ACE2
Binding assay
HiBIT tag
Mutation
Nano-luciferase

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