RESUMO
A comprehensive examination of protein-protein interactions (PPIs) is fundamental for the understanding of cellular machineries. However, limitations in current methodologies often prevent the detection of PPIs with low abundance proteins. To overcome this challenge, we develop a mRNA display with library of even-distribution (md-LED) method that facilitates the detection of low abundance binders with high specificity and sensitivity. As a proof-of-principle, we apply md-LED to IAV NS1 protein. Complementary to AP-MS, md-LED enables us to validate previously described PPIs as well as to identify novel NS1 interactors. We show that interacting with FASN allows NS1 to directly regulate the synthesis of cellular fatty acids. We also use md-LED to identify a mutant of NS1, D92Y, results in a loss of interaction with CPSF1. The use of high-throughput sequencing as the readout for md-LED enables sensitive quantification of interactions, ultimately enabling massively parallel experimentation for the investigation of PPIs.
