Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

Cell Rep. 2020 Oct 27;33(4):108322. doi: 10.1016/j.celrep.2020.108322. Epub 2020 Oct 12.


Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.

Keywords: COVID-19; HRV3C protease; antibody; biotinylated probe; coronavirus disease 2019; human rhinovirus 3C; single-chain Fc; structure-based design.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2
  • Antibodies, Neutralizing / immunology*
  • Antibodies, Viral / immunology*
  • Antibody Specificity / immunology
  • Binding Sites, Antibody / immunology
  • Biotinylation
  • COVID-19
  • Coronavirus Infections / immunology*
  • Cryoelectron Microscopy
  • Humans
  • Molecular Probes / immunology*
  • Pandemics
  • Peptidyl-Dipeptidase A / metabolism
  • Pneumonia, Viral / immunology*
  • Receptors, Virus / metabolism
  • Spike Glycoprotein, Coronavirus / immunology*


  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Molecular Probes
  • Receptors, Virus
  • Spike Glycoprotein, Coronavirus
  • Peptidyl-Dipeptidase A
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2