Figure 2

Optimised yeast display system for the screening of Nanobody libraries by cell sorting. (a) We designed novel vectors for the extracellular display of fusion proteins consisting of a Nanobody followed by Aga2p and by ACP. The Nanobody is fused at its C-terminus to Aga2p, leaving the CDRs fully exposed for antigen binding. (b) Enzymes such as Sfp Synthase can be used to covalently attach CoA derivatives containing fluorophores or biotin to a unique serine residue of the C-terminal ACP tag. (c) In our vectors, Nanobody libraries can be cloned as fusion proteins under the transcriptional control of the GAL1 promoter. The fusion protein is secreted by using the appS4 leader sequence. ACP can be replaced by other tags that are amenable to covalent orthogonal labelling of the displayed fusion protein: S6 (pNS6 vector) or SNAPf (pNSNAP vector).