title: A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions.
abstract: Low-affinity extracellular protein interactions are critical for cellular recognition processes, but existing methods to detect them are limited in scale, making genome-wide interaction screens technically challenging. To address this, we report here the miniaturization of the AVEXIS (avidity-based extracellular interaction screen) assay by using protein microarray technology. To achieve this, we have developed protein tags and sample preparation methods that enable the parallel purification of hundreds of recombinant proteins expressed in mammalian cells. We benchmarked the protein microarray-based assay against a set of known quantified receptor-ligand pairs and show that it is sensitive enough to detect even very weak interactions that are typical of this class of interactions. The increase in scale enables interaction screening against a dilution series of immobilized proteins on the microarray enabling the observation of saturation binding behaviors to show interaction specificity and also the estimation of interaction affinities directly from the primary screen. These methodological improvements now permit screening for novel extracellular receptor-ligand interactions on a genome-wide scale.
authors.affiliation: Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
authors.forename: Marcus
authors.initials: M
authors.lastname: Gallagher-Jones
authors.forename: Colin
authors.initials: C
authors.lastname: Barker
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Wright
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D003362:Cost-Benefit Analysis; D005110:Extracellular Space / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins; D008969:Molecular Sequence Data; D040081:Protein Array Analysis / Q000191:economics / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000191:economics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism; D046509:Tissue Culture Techniques; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000378:metabolism
chemical_list: D055767:Immobilized Proteins; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; D019809:Streptavidin