Camelid single-domain antibody fragments (‘nanobodies’) provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.
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Financial support for this work was provided by the Vallee Foundation (A.C.K.), the Smith Family Foundation (A.C.K.), National Institutes of Health grants 5DP5OD021345 (A.C.K.), 1DP5OD023048 (A.M.), and 1DP5OD023088 (A.M.R.), the Lundbeck Foundation (grant no. R37-A3457 to S.G.F.R.), and the Danish Independent Research Council (grant no. 0602-02407B to S.G.F.R.).
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(a–k) Randomly chosen nanobodies were expressed and purified from E. coli, then analyzed by size exclusion chromatography to assess monodispersity. (l) SDS-PAGE analysis of nanobody purity following one-step nickel affinity purification.
(a) The display system was engineered using the high affinity SIRPα variant CV1 as a test protein, and its ligand CD47 ectodomain as the staining reagent. A biotin tag is schematized as a glowing red circle. (b) Length of the stalk region determines accessibility of a displayed protein as a function of molecular weight. (c) Analytical flow cytometry plots showing length dependence for two staining reagents: CD47 biotin and α-HA antibody. The 649 amino acid long stalk was used in all nanobody display experiments.
(a) Library design was assessed by monitoring the change in amino acid frequency in CDR3 throughout selection rounds with HSA as the antigen. Few changes were observed, with the only notable trend a modest increase in basic residue frequency and a decline in acidic residue frequency. (b) Assessment of Nb.b201 binding to human serum albumin by surface plasmon resonance, comparison with mouse serum albumin which shows no detectable binding. (c) 2Fo-Fc composite omit map contoured at 1.5 σ for antigen bound Nb.b201. The structure of both bound (yellow) and free (gray) forms of the nanobody are shown, highlighting structural divergence. (d) 2Fo-Fc composite omit map contoured at 1.5 σ for free Nb.b201.
(a) Conditioned medium containing adiponectin (left lane) was used for selection of nanobodies. It shows a complex mixture of proteins as assessed by SDS-PAGE. For reference, purified adiponectin is shown in the right lane. Adiponectin exists as a mix of 16-mers, hexamers, and trimers. (b) Schematic of selection process. Fluorescent anti-FLAG antibody was used to specifically mark those yeast cells that display adiponectin-binding nanobodies. (c) Flow cytometry analysis of final clone pool, showing that the library was highly enriched in adiponectin-binding clones. (d) Sequences of five selected clones showed highly diverse CDR3 sequence composition and length. (e) Binding assessed using in vitro pull-down with purified adiponectin globular domain. (f) Binding to adiponectin was further confirmed in vitro using surface plasmon resonance. Kinetic fit is shown for clone Nb.AQ103.
On-yeast titration to estimate affinity of β2AR binding nanobodies. EC50 values are summarized in the lower right. Bottom panel shows measurement of conformational selectivity for selected clones as assessed by flow cytometry.