Baculovirus display for discovery of low‐affinity extracellular receptor‐ligand interactions using protein microarrays (595.2)

Abstract

When used in conjunction with multivalent baits, protein microarrays offer a robust technology for discovery of low‐affinity extracellular protein‐protein interactions (ePPIs). Baits for receptor‐matching screens generally consist of recombinantly expressed and purified soluble domains. Given that approximately two‐thirds of extracellular proteins are transmembrane‐containing proteins, it would be desirable to develop a system to express and display bait receptors in a native‐like membrane environment. Towards this end, we evaluated the use of baculovirus (BV) display as a platform for generating multivalent baits for protein microarray screens. Virion particles were generated displaying single‐transmembrane receptors BTLA, CD200 or EFNB2, representing a range of affinities for their interacting partners. Virions labeled with Cy5 fluorophore were screened against a microarray containing over 600 extracellular proteins and the results were compared to data derived from soluble Fc‐protein or bait‐coated protein A microbeads. An optimized protocol employing a blocking step with a non‐bait expressing control BV allowed the identification of the expected interactions with signal‐to‐noise similar or higher than obtained with the other formats. Our results demonstrate that BV display is suitable for detection of high‐ and low‐affinity ePPIs on protein microarrays. The platform eliminates the need for direct protein purification and provides a native‐like lipid environment for membrane‐associated receptors.