Date Published: May 01, 2012
Publisher: Academic Press
Author(s): Yi Sun, Marcus Gallagher-Jones, Colin Barker, Gavin J. Wright.
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.
Defining protein interaction networks on an increasingly large scale to understand how biological processes are coordinately regulated has been a focus of recent research interest. These efforts have relied on a small number of high-throughput assays such as biochemical purification followed by mass spectrometry [39,40], yeast two-hybrid screening [41,42], and other protein complementation assays . Although these approaches have revealed a great deal regarding the architecture of intracellular protein interaction networks, none of these assays is generally suitable for detecting low-affinity extracellular interactions. To address this problem, several scalable assays specifically designed to detect extracellular interactions have been developed [6,9,11–13,15] but are practically limited to screening modest library sizes of several hundred receptor proteins, whereas vertebrate genomes are known to contain several thousand . Here, we have shown that it is possible to implement the AVEXIS assay as a protein microarray format, which, together with improved sample handling and the development of custom-built protein sample preparation hardware, makes extracellular protein interaction screening feasible on a genome-wide scale.