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J Proteome Res. 2007 Jul;6(7):2587-95. Epub 2007 Jun 1.

A beta-galactosidase-based bacterial two-hybrid system to assess protein-protein interactions in the correct cellular environment.

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Department of Biochemistry, Physiology and Microbiology, Ghent University, Laboratory for Protein Biochemistry and Protein Engineering, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.


The vast majority of proteins functions in complex with one or more of the same or other proteins, indicating that protein-protein interactions play crucial roles in biology. Here, we present a beta-galactosidase reconstitution-based bacterial two-hybrid system in which two proteins of interest are fused to two non-functional but complementing beta-galactosidase truncations (Delta alpha and Delta omega). The level of complemented beta-galactosidase activity, driven by the protein-protein recognition between both non-beta-galactosidase parts of the chimeras, reflects whether or not the proteins of interest interact. Our approach was validated by reconfirming some well-established Escherichia coli cytoplasmic and membranous interactions, including well-chosen mutants, and providing the first in vivo evidence for the transient periplasmic interaction between Rhodobacter capsulatus cytochrome c2 and cytochrome c peroxidase. We demonstrated the major advantages of this in vivo two-hybrid technique: i) analyses of interactions are not limited to particular cellular compartments, ii) the potential of using the system in mutation-driven structure-function studies, and iii) the possibility of its application to transiently interacting proteins. These benefits demonstrate the relevance of the method as a powerful new tool in the broad spectrum of interaction assessment methods.

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