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Nucleic Acids Research 2005 33(2):e18; doi:10.1093/nar/gni011
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Published online 1 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Novel mobilizable prokaryotic two-hybrid system vectors for high-throughput protein interaction mapping in Escherichia coli by bacterial conjugation

Paul Clarke, Páraic Ó Cuív and Michael O'Connell*

School Of Biotechnology, Dublin City University Glasnevin, Dublin 9, Ireland

*To whom correspondence should be addressed. Tel: +353 1 7005318; Fax: +353 1 7005412; Email: michael.oconnell{at}dcu.ie

Received November 5, 2004. Revised December 20, 2004. Accepted December 20, 2004.

Since its initial description, the yeast two-hybrid (Y2H) system has been widely used for the detection and analysis of protein–protein interactions. Mating-based strategies have been developed permitting its application for automated proteomic interaction mapping projects using both exhaustive and high-throughput strategies. More recently, a number of prokaryotic two-hybrid (P2H) systems have been developed but, despite the many advantages such Escherichia coli-based systems have over the Y2H system, they have not yet been widely implemented for proteomic interaction mapping. This may be largely due to the fact that high-throughput strategies employing bacterial transformation are not as amenable to automation as Y2H mating-based strategies. Here, we describe the construction of novel conjugative P2H system vectors. These vectors carry a mobilization element of the IncP{alpha} group plasmid RP4 and can therefore be mobilized with high efficiency from an E.coli donor strain encoding all of the required transport functions in trans. We demonstrate how these vectors permit the exploitation of bacterial conjugation for technically simplified and automated proteomic interaction mapping strategies in E.coli, analogous to the mating-based strategies developed for the Y2H system.


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