Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes
Adam S. Olia,
Phinikoula S. Katsamba,
Bailey B. Banach,
Ahmed S. Fahad,
Sheila N. Lopez Acevedo,
Matheus Oliveira de Souza,
Jacy R. Wolfe,
David D Ho,
Kizzmekia S. Corbett,
Brandon J. DeKosky,
John R. Mascola,
Nancy J. Sullivan,
Peter D. Kwong
Posted 23 Jun 2020
bioRxiv DOI: 10.1101/2020.06.22.166033 (published DOI: 10.1016/j.celrep.2020.108322)
Posted 23 Jun 2020
Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed 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 included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized 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. ### Competing Interest Statement The authors have declared no competing interest.
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