title: 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.
authors: Department of Protein Chemistry, Genentech, South San Francisco, CA 94080, USA.
authors: Irene
authors: I
authors: Tom
authors: Department of Structural Biology, Genentech, South San Francisco, CA 94080, USA.
authors: Alberto
authors: A
authors: Estevez
authors: Department of Structural Biology, Genentech, South San Francisco, CA 94080, USA.
authors: Krista
authors: K
authors: Bowman
authors: Department of Protein Chemistry, Genentech, South San Francisco, CA 94080, USA. Electronic address: gonzal29@gene.com.
authors: Lino C
authors: LC
authors: Gonzalez
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D036388:Ephrin-B2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D059629:Signal-To-Noise Ratio; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D036388:Ephrin-B2; D008024:Ligands; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C042739:antigens, CD200
keywords: Baculovirus display; Low-affinity interactions; Protein microarray; Receptor–ligand interactions