A Multireporter Bacterial 2‑Hybrid Assay for the High-Throughput and Dynamic Assay of PDZ Domain–Peptide Interactions

• Published in: ACS synthetic biology Vol. 8; no. 5; pp. 918 - 928
• Main Authors: Ichikawa, David M, Corbi-Verge, Carles, Shen, Michael J, Snider, Jamie, Wong, Victoria, Stagljar, Igor, Kim, Philip M, Noyes, Marcus B
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17-05-2019
• Subjects: Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - metabolism; Amino Acid Sequence; Escherichia coli - metabolism; Genes, Reporter; High-Throughput Screening Assays - methods; Humans; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; PDZ Domains; Peptide Library; Peptides - chemistry; Peptides - metabolism; Protein Binding; synthetic biology; protein interaction; PDZ domain; bacteria hybrid assay
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.8b00499

The accurate determination of protein–protein interactions has been an important focus of molecular biology toward 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 screening of large peptide libraries followed by the sorting 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.