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Molecular & Cellular Proteomics 4:819-826, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

A Combined Yeast/Bacteria Two-hybrid System

Development and Evaluation^*

Ilya G. Serebriiskii^,, Rui Fang^¶, Ekaterina Latypova, Richard Hopkins^||,**, Charles Vinson, J. Keith Joung^¶ and Erica A. Golemis

From the Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, ^¶ Molecular Pathology Unit, Department of Pathology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, ^|| Telethon Institute for Child Health Research, West Perth, WA 6872 Australia, ^** Phylogica, Ltd., West Perth, WA 6872 Australia, and Division of Basic Science, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.

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