NDLI: High-precision mapping of protein–protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform

Content Provider	PubMed Central
Author	Pajunen, Maria Turakainen, Hilkka Poussu, Eini Johan, Peränen Vihinen, Mauno Savilahti, Harri
Copyright Year	2007
Abstract	Understanding networks of protein–protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein–protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1.
Related Links	http://dx.doi.org/10.1093/nar/gkm563
Starting Page	103
File Format	PDF
ISSN	03051048
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	16
Volume Number	35
Language	English
Publisher	Oxford University Press
Publisher Date	2007-08-01
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Genetics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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High-precision mapping of protein–protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform

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

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Yeast two-hybrid system for studying protein-protein interactions--stage 2: Transforming and characterizing the library.

High-precision mapping of protein–protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform

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