A beta-galactosidase-based bacterial two-hybrid system to assess protein-protein interactions in the correct cellular environment.
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.
Borloo
Jimmy
J
Department of Biochemistry, Physiology and Microbiology, Ghent University, Laboratory for Protein Biochemistry and Protein Engineering, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium. Jimmy.Borloo@ugent.be
De Smet
Lina
L
Vergauwen
Bjorn
B
Van Beeumen
Jozef J
JJ
Devreese
Bart
B
D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045362:Cytochromes c2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000201:enzymology; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D045362:Cytochromes c2; D003578:Cytochrome-c Peroxidase; D001616:beta-Galactosidase