A multi-reporter bacterial 2-hybrid assay for the fast, simple, and dynamic assay of PDZ domain – peptide interactions

Abstract

The accurate determination of protein-protein interactions is an important goal of molecular biology and much progress has been made with modern methods emerging in the past decade. However, current methods have limitations, including scale and restriction to high affinity interactions. This has limited our understanding of the large subset of protein-motif interactions. Here we describe a modified bacterial-hybrid assay that employs a combined selectable and scalable reporter system that allows the screen of large protein-peptide libraries and their sort by relative strength. 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. Corresponding Author of the project. D.M.I. and M.J.S. carried out the bacterial experiments while C.C.V. executed all computational analysis. J.S. and V.W. carried out all MaMTH construct generation and assays. I.S. oversaw all MaMTH assays and provided critical analysis of results. D.M.I., M.B.N. and P.M.K. wrote the manuscript.