title: Application of Mu in vitro transposition for high-precision mapping of protein-protein interfaces on a yeast two-hybrid platform.
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. Transposon-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.
authors.affiliation: Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland.
authors.forename: Maria
authors.initials: M
authors.lastname: Pajunen
authors.forename: Eini
authors.initials: E
authors.lastname: Poussu
authors.forename: Hilkka
authors.initials: H
authors.lastname: Turakainen
authors.forename: Harri
authors.initials: H
authors.lastname: Savilahti
mesh_terms: D004251:DNA Transposable Elements / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods; D010957:Plasmids; D023302:Protein Footprinting; D025941:Protein Interaction Mapping / Q000379:methods*; D019895:Transposases / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D004251:DNA Transposable Elements; C114697:mu transposase; D019895:Transposases