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

Zhou, T.; Teng, I.-T.; Olia, A. S.; Cerutti, G.; Gorman, J.; Nazzari, A.; Shi, W.; Tsybovsky, Y.; Wang, L.; Wang, S.; Zhang, B.; Zhang, Y.; Katsamba, P. S.; Petrova, Y.; Banach, B. B.; Fahad, A. S.; Liu, L.; Lopez Acevedo, S. N.; Madan, B.; Oliveira de Souza, M.; Pan, X.; Wang, P.; Wolfe, J. R.; Yin, M.; Ho, D. D.; Phung, E.; DiPiazza, A.; Chang, L.; Abiona, O.; Corbett, K. S.; DeKosky, B. J.; Graham, B. S.; Mascola, J. R.; Misasi, J.; Ruckwardt, T.; Sullivan, N. J.; Shapiro, L.; Kwong, P. D.

doi:10.1101/2020.06.22.166033

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bioRxiv · 10.1101/2020.06.22.166033

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

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Abstract

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.

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Zhou, T., Teng, I.-T., Olia, A. S., Cerutti, G., Gorman, J., Nazzari, A., Shi, W., Tsybovsky, Y., Wang, L., Wang, S., Zhang, B., Zhang, Y., Katsamba, P. S., Petrova, Y., Banach, B. B., Fahad, A. S., Liu, L., Lopez Acevedo, S. N., Madan, B., Oliveira de Souza, M., Pan, X., Wang, P., Wolfe, J. R., Yin, M., Ho, D. D., Phung, E., DiPiazza, A., Chang, L., Abiona, O., Corbett, K. S., DeKosky, B. J., Graham, B. S., Mascola, J. R., Misasi, J., Ruckwardt, T., Sullivan, N. J., Shapiro, L., Kwong, P. D.. 2020-06-23. Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes. https://doi.org/10.1101/2020.06.22.166033

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