Application of Mu in vitro transposition for high-precision mapping of protein-protein interfaces on a yeast two-hybrid platform

: Pajunen M, Poussu E, Turakainen H, Savilahti H

Publisher: ACADEMIC PRESS INC ELSEVIER SCIENCE

: 2009

Methods

METHODS

: METHODS

: 49

: 3

: 255

: 262

: 8

: 1046-2023

DOI: https://doi.org/10.1016/j.ymeth.2009.04.014

High-precision mapping of regions involved in protein-protein interfaces of interacting protein partners is an essential component on a path to understand various cellular functions. Trans poson-based systems, particularly those involving in vitro reactions, offer exhaustive insertion mutant libraries and high-throughput platforms for many types of genetic analyses. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision that is based on transposition-assisted construction, sampling, and analysis of a comprehensive insertion mutant library. The methodology integrates random pentapeptide mutagenesis of proteins, yeast two-hybrid screening, and high-resolution genetic footprinting. This straightforward strategy is general, and it provides a rapid and easy means to identify critical contact regions in proteins without the requirement of prior structural knowledge. (C) 2009 Elsevier Inc. All rights reserved.