Identifying and optimizing intracellular protein-protein interactions using bacterial genetic selection
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Author list: Waraho D., Delisa M.P.
Publisher: Humana Press
Publication year: 2012
Journal: Methods in Molecular Biology (1064-3745)
Volume number: 813
Start page: 125
End page: 143
Number of pages: 19
ISBN: 9781617794117
ISSN: 1064-3745
Languages: English-Great Britain (EN-GB)
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Abstract
Protein-protein interactions are crucial for the vast majority of biological processes. To fully understand these processes therefore requires methods for identifying protein interactions within the complex cellular environment. To isolate interacting proteins, we have developed a simple and reliable genetic selection by exploiting the inbuilt "hitchhiker" mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method is based on the unique ability of the Tat system to efficiently co-localize noncovalent complexes of two folded polypeptides to the periplasmic space of E. coli. The genetic selection is comprised of two engineered fusion proteins: an N-terminal Tat signal peptide fused to the protein of interest, and the known or putative partner protein fused to mature TEM-1 β-lactamase. The efficiency with which co-localized β-lactamase chimeras are exported in the periplasm, and thus confer ampicillin resistance to cells, is directly linked to the relative binding affinity of the protein-ligand system. Thus, ampicillin resistance can be used as a convenient readout for identifying and optimizing protein interactions in E. coli. Furthermore, because Tat substrates must be correctly folded for export, our method favors the identification of soluble, non-aggregating, protease-resistant protein pairs. Overall, we anticipate that this new selection tool will be useful for discovering and engineering protein drugs, protein complexes for structural biology, factors that inhibit PPIs, and components for synthetic biology. © 2012 Springer Science+Business Media, LLC.
Keywords
Affi nity maturation, Antibody engineering, Bacterial 2-hybrid system, Genetic selection, Intracellular antibodies, Proteinprotein interaction, Tat pathway
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