Baculovirus display for discovery of low-affinity extracellular receptor–ligand interactions using protein microarrays

Abstract

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

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions

“…Yeast and mammalian two-hybrid systems use libraries that direct fusion protein expression intracellularly and are therefore not readily applicable to extracellular domains (ECDs), which generally do not fold correctly in the cytoplasm ( Fiebitz et al, 2008 ; Rajagopala, 2015 ). Direct binding assays using immobilized fusion proteins have high sensitivity but require production, purification, and analysis of hundreds or thousands of proteins ( Bushell et al, 2008 ; Ramani et al, 2012 ; Özkan et al, 2013 ; Tom et al, 2015 ; Visser et al, 2015 ; Hsu et al, 2017 ). Identifying PPIs based on a bioassay has high specificity and biological relevance but requires some knowledge of protein function (e.g.…”