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

Zhou, Tongqing; Teng, I-Ting; Olia, Adam S.; Cerutti, Gabriele; Gorman, Jason; Nazzari, Alexandra; Shi, Wei; Tsybovsky, Yaroslav; Wang, Lingshu; Wang, Shuishu; Zhang, Baoshan; Zhang, Yi; Katsamba, Phinikoula S.; Petrova, Yuliya; Banach, Bailey B.; Fahad, Ahmed S.; Liu, Lihong; Lopez Acevedo, Sheila N.; Madan, Bharat; de Souza, Matheus Oliveira; Pan, Xiaoli; Wang, Pengfei; Wolfe, Jacy R.; Yin, Michael; Ho, David D.; Phung, Emily; DiPiazza, Anthony; Chang, Lauren; Abiona, Olubukula; Corbett, Kizzmekia S.; DeKosky, Brandon J.; Graham, Barney S.; Mascola, John R.; Misasi, John; Ruckwardt, Tracy; Sullivan, Nancy J.; Shapiro, Lawrence; Kwong, Peter D.

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.