Design and characterization of SpyTagged HAs, SpyCatcher VLPs, and SpyCatcher-NPs.
A. Top: Schematic of the SpyTagged HA construct (SP = signal peptide). The HA2 ectodomain is followed by a foldon trimerization domain from T4 fibritin, a 13-residue SpyTag, and a 6x-His tag. Amino acids are numbered according to the H3 nomenclature. Horizontal lines represent Gly4Ser linkers. Bottom: Surface representation of an HA trimer structure (PDB 3VUN), schematic of a SpyTagged HA, and list of influenza strains from which SpyTagged HAs were derived. B. SEC profiles and reducing SDS-PAGE analysis of 8 purified SpyTagged HAs that contain Y98F substitution. C. SpyCatcher-AP205 VLPs. Top: Schematic of construct. Bottom: EM structure of T = 3 AP205 particle (PDB 5FS3) [23] with the locations of SpyCatcher fusion sites indicated by red dots (left) and schematic SpyCatcher-VLP (right). D. Purification of SpyCatcher-VLPs. Left: SEC profile with peak representing properly-assembled VLPs indicated. Right: Reducing and non-reducing SDS-PAGE of two fractions corresponding to the VLP fractions in red on the SEC trace. E. SpyCatcher-mi3 NPs. Top: schematic of construct. Bottom: Cryo-EM structure of I3-01 particle related to mi3 [24] with the locations of SpyCatcher fusion sites indicated by red dots (left) and schematic SpyCatcher-NP (right). F. Purification of SpyCatcher-NPs. Left: SEC profile. Right: Reducing SDS-PAGE of fractions corresponding to the mi3 fractions shaded in red on the SEC trace.
