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Nucleic Acids Research Advance Access published online on August 15, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm563
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Maria Pajunen1, Hilkka Turakainen1, Eini Poussu1, Johan Peränen1, Mauno Vihinen2,3 and Harri Savilahti1,4,*

1Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, 2Institute of Medical Technology, University of Tampere, 3Research Unit, Tampere University Hospital, Tampere and 4Division of Genetics and Physiology, Department of Biology, University of Turku, Finland

*To whom correspondence should be addressed. Tel: +358 9 191 59516; Fax: +358 9 191 59366; Email: harri.savilahti{at}helsinki.fi

Received May 21, 2007. Revised June 28, 2007. Accepted July 6, 2007.

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.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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