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

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.
Mesh Terms:
Angiotensin-Converting Enzyme 2, Antibodies, Neutralizing, Antibodies, Viral, Antibody Specificity, Binding Sites, Antibody, Biotinylation, COVID-19, Coronavirus Infections, Cryoelectron Microscopy, Humans, Molecular Probes, Pandemics, Peptidyl-Dipeptidase A, Pneumonia, Viral, Receptors, Virus, Spike Glycoprotein, Coronavirus
Cell Rep
Date: Oct. 27, 2020
Download Curated Data For This Publication
250678
Switch View:
  • Interactions 2