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

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

Research output: Contribution to journalArticlepeer-review

41 Scopus citations


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 ∼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.

Original languageEnglish
Article number108322
JournalCell Reports
Issue number4
StatePublished - 27 Oct 2020
Externally publishedYes


  • COVID-19
  • HRV3C protease
  • antibody
  • biotinylated probe
  • coronavirus disease 2019
  • human rhinovirus 3C
  • single-chain Fc
  • structure-based design


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