A1 Refereed original research article in a scientific journal
Application of Mu in vitro transposition for high-precision mapping of protein-protein interfaces on a yeast two-hybrid platform
Authors: Pajunen M, Poussu E, Turakainen H, Savilahti H
Publisher: ACADEMIC PRESS INC ELSEVIER SCIENCE
Publication year: 2009
Journal:Methods
Journal name in sourceMETHODS
Journal acronym: METHODS
Volume: 49
Issue: 3
First page : 255
Last page: 262
Number of pages: 8
ISSN: 1046-2023
DOI: https://doi.org/10.1016/j.ymeth.2009.04.014
Abstract
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.
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.
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