A multi-reporter bacterial 2-hybrid assay for the high-throughput and dynamic assay of PDZ domain - peptide interactions.

The accurate determination of protein-protein interactions has been an important focus of molecular biology towards which much progress has been made due to the continuous development of existing and new technologies. However, current methods can have limitations, including scale and restriction to high affinity interactions, limiting our understanding of a large subset of these interactions. Here we describe a modified bacterial-hybrid assay that employs combined selectable and scalable reporters that enable the sensitive screen of large peptide libraries followed by the sort of positive interactions by the level of reporter output. We have applied this tool to characterize a set of human and E. coli PDZ domains. Our results are consistent with prior characterization of these proteins and the improved sensitivity increases our ability to predict known and novel in vivo binding partners. This approach allows for the recovery of a wide range of affinities with a high throughput method that does not sacrifice the scale of the screen.