Nat. Struct. Mol. Biol. 25, 289–296 (2018)

Nanobodies have found widespread use in structural biology, in cell biology and as therapeutics, but almost all are still derived from alpaca and llama immunizations, which is time consuming and expensive. To overcome these limitations, McMahon et al. developed a surface-display platform for the isolation of selective nanobodies from yeast cells. The authors first generated a synthetic nanobody library based on sequence alignments of structurally characterized nanobodies. The designed nanobodies were then fused to a long linker region and a C-terminal anchor sequence that tethers them to the yeast cell wall for surface display while the target protein was labeled with a fluorescent dye. Yeast cells expressing the nanobodies were then incubated with the labeled protein, which was followed by repeated selection rounds with anti-fluorophore magnetic microbeads or fluorescence-activated cell sorting. Single yeast colonies were sequenced after the final selection round. The identified nanobodies were then expressed and purified from Escherichia coli. The authors demonstrate that conformationally selective G-protein-coupled receptor-binding nanobodies can be isolated, which is useful for functional studies and crystallization. The method is versatile, because either purified or nonpurified antigens can be used, and other selection procedures may also be implemented. This rapid nanobody discovery platform could make nanobody preparation more easily accessible and cost-effective.