RT Journal Article SR Electronic T1 Platform for rapid nanobody discovery in vitro JF bioRxiv FD Cold Spring Harbor Laboratory SP 151043 DO 10.1101/151043 A1 Conor McMahon A1 Alexander S. Baier A1 Sanduo Zheng A1 Roberta Pascolutti A1 Janice X. Ong A1 Sarah C. Erlandson A1 Daniel Hilger A1 Aaron M. Ring A1 Aashish Manglik A1 Andrew C. Kruse YR 2017 UL http://biorxiv.org/content/early/2017/06/16/151043.abstract AB Camelid single-domain antibody fragments (“nanobodies”) provide the remarkable specificity of antibodies within a single immunoglobulin VHH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available.