Nanoscale spherical-supported membranes as novel platforms for improving the phage display screening of antibody mimetics against membrane protein targets

Membrane proteins represent the majority of therapeutic targets for the antibody-based drugs available today. These are routinely identified via phage display screening, but traditional antigen presentation methods require membrane protein targets to be detergent-solubilised in order to preserve their native conformations post-purification. Unfortunately, detergent solubilisation can not only lead to gradual target denaturation over time, but the detergent micelles can also occlude important epitopes on the extramembranous loops and thus prevent the discovery of antibody binders. The current thesis aimed to demonstrate that, by reconstituting purified membrane proteins into spherical-supported bilayer lipid membranes (SSBLMs) deposited on nanosized substrates, a versatile platform can be constructed for performing phage display screening against membrane protein targets, while not only presenting these within a native-like lipid environment, but also eliminating detergents from the screening phase altogether. For providing proof-of-concept, 100- and 200 nm silica nanoparticles were covered with POPC SSBLMs embedding the bacterial nucleoside transporter NupC. Full substrate coverage and the correct formation of the lipid bilayer components were established via spectrofluorometry, using fluorescent labelling and small-angle X-ray scattering (SAXS) respectively, while Western blotting and high-affinity antibody binding confirmed the presence of SSBLM-embedded NupC. The platform was then used to screen designed ankyrin repeat proteins (DARPins) against a His6-tagged construct of NupC across different screening formats so as to offer a comparison to the classic 96-well plate antigen presentation method. Following that, the DARPin binders showing the highest potential affinity for NupC were purified and subjected to further binding validation assays against two other constructs – detergent-solubilised double Strep-tagged NupC and SSBLM-embedded untagged/wild-type NupC – in order to identify any binders targeting extramembranous epitopes that would be accessible in vivo as well. Ultimately, the results presented throughout indicated that SSBLMs constitute a promising means of screening antibody binders against membrane protein targets embedded in a close-to-native format.