High-resolution protein-protein interaction mapping using all-versus-all sequencing (AVA-Seq). Academic Article uri icon

Overview

abstract

  • Two-hybrid systems can be used for investigating protein-protein interactions and may provide important information about gene products with unknown function. Despite their success in mapping protein interactions, two-hybrid systems have remained mostly untouched by improvements in next-generation DNA sequencing. The two-hybrid systems rely on one-versus-all methods in which each bait is sequentially screened against an entire library. Here, we developed a screening method that joins both bait and prey as a convergent fusion into one bacterial plasmid vector that can then be amplified and paired-end sequencing by next-generation sequencing (NGS). Our method enables all-versus-all sequencing (AVA-Seq) and utilizes NGS to remove multiple bottlenecks of the two-hybrid system. AVA-Seq allows for high-resolution protein-protein interaction mapping of a small set of proteins and has the potential for lower-resolution mapping of entire proteomes. Features of the system include ORF selection to improve efficiency, high bacterial transformation efficiency, a convergent fusion vector to allow paired-end sequencing of interactors, and the use of protein fragments rather than full-length proteins to better resolve specific protein contact points. We demonstrate the system's strengths and limitations on a set of proteins known to interact in humans and provide a framework for future large-scale projects.

publication date

  • June 10, 2019

Research

keywords

  • Protein Interaction Mapping

Identity

PubMed Central ID

  • PMC6663860

Scopus Document Identifier

  • 85070100024

Digital Object Identifier (DOI)

  • 10.1126/science.7878469

PubMed ID

  • 31182485

Additional Document Info

volume

  • 294

issue

  • 30