A -Galactosidase-Based Bacterial Two-Hybrid System To Assess Protein-Protein Interactions in the Correct Cellular Env
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- 作者：Jimmy Borloo ; Lina De Smet ; Bjorn Vergauwen ; Jozef J. Van Beeumen ; Bart Devreese
- 刊名：Journal of Proteome Research
- 出版年：2007
- 出版时间：July 2007
- 年：2007
- 卷：6
- 期：7
- 页码：2587 - 2595
- 全文大小：299K
- 年卷期：v.6,no.7(July 2007)
- ISSN：1535-3907
文摘
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 
-galactosidase reconstitution-based bacterial two-hybrid system in which two proteins of interest are fusedto two non-functional but complementing -galactosidase truncations (
and 
). The level ofcomplemented -galactosidase activity, driven by the protein-protein recognition between both non--galactosidase parts of the chimeras, reflects whether or not the proteins of interest interact. Ourapproach was validated by reconfirming some well-established Escherichia coli cytoplasmic andmembranous interactions, including well-chosen mutants, and providing the first in vivo evidence forthe transient periplasmic interaction between Rhodobacter capsulatus cytochrome c2 and cytochromec peroxidase. We demonstrated the major advantages of this in vivo two-hybrid technique: i) analysesof interactions are not limited to particular cellular compartments, ii) the potential of using the systemin mutation-driven structure-function studies, and iii) the possibility of its application to transientlyinteracting proteins. These benefits demonstrate the relevance of the method as a powerful new toolin the broad spectrum of interaction assessment methods.
-galactosidase reconstitution-based bacterial two-hybrid system in which two proteins of interest are fusedto two non-functional but complementing -galactosidase truncations ( and ). The level ofcomplemented -galactosidase activity, driven by the protein-protein recognition between both non--galactosidase parts of the chimeras, reflects whether or not the proteins of interest interact. Ourapproach was validated by reconfirming some well-established Escherichia coli cytoplasmic andmembranous interactions, including well-chosen mutants, and providing the first in vivo evidence forthe transient periplasmic interaction between Rhodobacter capsulatus cytochrome c2 and cytochromec peroxidase. We demonstrated the major advantages of this in vivo two-hybrid technique: i) analysesof interactions are not limited to particular cellular compartments, ii) the potential of using the systemin mutation-driven structure-function studies, and iii) the possibility of its application to transientlyinteracting proteins. These benefits demonstrate the relevance of the method as a powerful new toolin the broad spectrum of interaction assessment methods.