TY - JOUR T1 - Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes JF - bioRxiv DO - 10.1101/2020.06.22.166033 SP - 2020.06.22.166033 AU - Tongqing Zhou AU - I-Ting Teng AU - Adam S. Olia AU - Gabriele Cerutti AU - Jason Gorman AU - Alexandra Nazzari AU - Wei Shi AU - Yaroslav Tsybovsky AU - Lingshu Wang AU - Shuishu Wang AU - Baoshan Zhang AU - Yi Zhang AU - Phinikoula S. Katsamba AU - Yuliya Petrova AU - Bailey B. Banach AU - Ahmed S. Fahad AU - Lihong Liu AU - Sheila N. Lopez Acevedo AU - Bharat Madan AU - Matheus Oliveira de Souza AU - Xiaoli Pan AU - Pengfei Wang AU - Jacy R. Wolfe AU - Michael Yin AU - David D. Ho AU - Emily Phung AU - Anthony DiPiazza AU - Lauren Chang AU - Olubukula Abiona AU - Kizzmekia S. Corbett AU - Brandon J. DeKosky AU - Barney S. Graham AU - John R. Mascola AU - John Misasi AU - Tracy Ruckwardt AU - Nancy J. Sullivan AU - Lawrence Shapiro AU - Peter D. Kwong Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/06/23/2020.06.22.166033.abstract N2 - 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 StatementThe authors have declared no competing interest. ER -