Application of Mu in vitro transposition for high-precision mapping of protein-protein interfaces on a yeast two-hybrid platform.
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.
Pajunen
Maria
M
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland.
Poussu
Eini
E
Turakainen
Hilkka
H
Savilahti
Harri
H
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
D004251:DNA Transposable Elements; C114697:mu transposase; D019895:Transposases