Site-specific analysis of the SARS-CoV-2 glycan shield

Yasunori Watanabe; Joel D. Allen; Daniel Wrapp; Jason S. McLellan; Max Crispin

To resolve the site-specific glycosylation of SARS-CoV-2 S protein and visualize the distribution of glycoforms across the protein surface, we expressed and purified recombinant soluble material in an identical manner to that which was used to obtain the high-resolution cryo-electron microscopy (cryo-EM) structure, albeit without glycan processing blockade using kifunensine 18 . This soluble recombinant variant of the S protein contains all 22 glycans on the SARS-CoV-2 S protein ( Figure 1A) . Stabilization of the trimeric prefusion structure was achieved using the "2P" stabilizing mutations 24 at residues 986 and 987 in addition to a Cterminal trimerization motif. This ensures that the quaternary structure remains intact during glycan processing, as in the case of HIV Env mimetics, this is known to influence glycosylation of certain sites 16, 25 . Prior to analysis, supernatant containing the recombinant SARS-CoV-2 was purified using a C-terminal StrepTag followed by size-exclusion chromatography to ensure only native-like trimeric SARS-CoV-2 S protein is analyzed ( Figure 1B) . The trimeric conformation of the purified material was validated using negative stain electron microscopy ( Figure 1C ).