Figure 1
Production and functional evaluation of mammalian extracellular antigen-SpyCatcher fusions. (a) Schematic representation of the direct capture and immobilization (dCI) of SpyC-antigens via surface-bound bSpyT. (b,c) Mammalian cell expression and direct capture of representative SpyC-antigen fusions (see Table 1) on bSpyT-loaded magnetic beads. (b) Coomassie-stained SDS-PAGE (reducing). The insert-less pSTEVe49 vector cassette expresses the free HA-tagged SpyC (far right). (c) Anti-HA tag ECL Western staining of recombinant SpyC-antigens expressed in HEK culture supernatants (‘S’), and following selective covalent enrichment on SpyT beads (‘B’). Covalent immobilization to SpyT on the beads results in a shift to a higher MW corresponding to the mass of the peptide adduct. Samples were run on separate gels and interspersed with irrelevant samples, but were processed in parallel. Full-length gel and blot images can be found in Supplementary Fig. S2. (c) ELISA illustrating functional recognition of selected immobilized dCI SpyC-antigens by cognate recombinant ligands.
