Expression and Purification of SARS-related Spike Glycoproteins for Cryo-EM Analysis

Coronaviridae spike glycoproteins mediate viral entry and fusion to host cells through binding to host receptors (i.e., ACE2, DPP4) and are key components in determining viral host range, making them targets for antiviral research. Here, we describe the expression, purification, and characterization of recombinant spike proteins to aid in protein characterization and analysis. These protocols were used for the production of spike glycoproteins from civet, pangolin, and bat coronaviruses, as well as high-resolution cryo-electron microscopy (cryo-EM) structural analysis of bat and civet host coronavirus spike glycoproteins (Hills et al., 2024). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of SARS-CoV spike protein from ExpiCHO cells Basic Protocol 2: Preparation of SARS-CoV spike protein for visualization by negative-stain transmission electron microscopy and cryo-electron microscopy.