Journal
CELL REPORTS
Volume 33, Issue 4, Pages -Publisher
CELL PRESS
DOI: 10.1016/j.celrep.2020.108322
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Funding
- Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID)
- COVID-19 Fast Grants
- Jack Ma Foundation
- Self Graduate Fellowship Program
- NIH [DP5OD023118, R21AI143407, R21AI144408]
- Simons Foundation [SF349247]
- NYSTAR
- NIH National Institute of General Medical Sciences [GM103310]
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [ZIAAI005149, ZIAAI005147, ZIAAI005125, ZIAAI005022] Funding Source: NIH RePORTER
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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 similar to 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.
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