Synthetic Single Domain Antibodies for the Conformational Trapping of Membrane Proteins

Zimmermann I, et al., doi:, bioRxiv, 2017

The group developed a novel in vitro selection platform, which builds on synthetic nanobodies called sybodies. Inspired by the shape diversity of natural nanobodies, three sybody libraries exhibiting different randomized surface shapes were engineered for high thermal stability. The conformation-selective, high affinity sybodies were engineered against the human glycine transporter GlyT1, the human equilibrative nucleotide transporter ENT1 and a bacterial ABC transporter. This platform is built exclusively on commercially available reagents and enables nonspecialized labs to generate conformation-specific binders against previously intractable protein targets. The Octet RED96 System was applied for competition measurements. Streptavidin sensors were loaded with biotinylated MBP and then dipped in wells containing Sb_MBP#1 which leads to the formation of the Sybody-MBP complex. Sensors containing the complex were sequentially dipped in a row of wells containing Sb_MBP#1 and increasing concentrations of maltose (0.1, 1, 10, 100, 1000 μM). The reversibility of the competition was shown by decreasing maltose concentrations (1000, 100, 10, 1, 0.1, 0 μM) again in the presence of Sybody Sb_MBP#1. BLI measurements were conducted for biophysical analysis of sybody-MBP interactions.

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