Figure 6

Orthogonally labelled Nanobodies can be functionally shaved from yeast cells and immobilised. (a) Nanobodies can be displayed on the yeast surface as Aga2p-ACP fusions and orthogonally biotinylated via ACP by adding a CoA-biotin derivative in the presence of Sfp synthase. Subsequent, this biotinylated Nanobody-Aga2p-ACP fusion protein can efficiently be shaved from the yeast surface by DTT and immobilised on streptavidin coated (SA) materials for other applications including BLI. (b) Binding isotherms (raw data) of the immobilisation of a biotinylated Nanobody-Aga2p-ACP fusion protein and the subsequent binding of its antigen to an SA-coated biosensor measured by BLI on OctetRED96. A 10 ml culture of yeast, displaying Nanobody MP1031_B7 as an Nb-Aga2p-ACP fusion was resuspended in with CoA-biotin and Sfp synthase and shaved by adding 2 mM DTT. A SA biosensor (ForteBio) was dipped into the supernatant (step 1) and equilibrated into buffer (step 2). Next, the biosensor was incubated with different FIXa concentrations to follow the association (step 3) and dissociation (step 4) of the antigen.