High resolution protein-protein interaction mapping using all-versus-all sequencing (AVA-seq)

Two-hybrid systems test for protein-protein interactions and can provide important information for genes with unknown function. Despite their success, two-hybrid systems have remained mostly untouched by improvements from next-generation DNA sequencing. Here we present a method for all-versus-all pr...

Full description

Saved in:
Bibliographic Details
Published inbioRxiv
Main Authors Andrews, Simeon S, Ramadan, Stephanie, Al-Thani, Nayra M, Ikhlak Ahmed, Mohamoud, Yasmin A, Malek, Joel
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 05.11.2018
Subjects
DNA sequencing
Genetic transformation
Next-generation sequencing
Peptide mapping
Protein interaction
Proteins
Online AccessGet full text

Cover

More Information
Summary:Two-hybrid systems test for protein-protein interactions and can provide important information for genes with unknown function. Despite their success, two-hybrid systems have remained mostly untouched by improvements from next-generation DNA sequencing. Here we present a method for all-versus-all protein interaction mapping (AVA-seq) that utilizes next-generation sequencing to remove multiple bottlenecks of the two-hybrid process. The method allows for high resolution protein-protein interaction mapping of a small set of proteins, or the potential for lower-resolution mapping of entire proteomes. Features of the system include open-reading frame 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 genes 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.
DOI:10.1101/462309