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PLoS One. 2010 Nov 2;5(11):e13785. doi: 10.1371/journal.pone.0013785.

IC-tagging and protein relocation to ARV muNS inclusions: a method to study protein-protein interactions in the cytoplasm or nucleus of living cells.

Author information

1
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Center for Research in Biological Chemistry and Molecular Materials, University of Santiago de Compostela, Santiago de Compostela, Spain.

Abstract

BACKGROUND:

Characterization of protein-protein interactions is essential for understanding cellular functions. Although there are many published methods to analyze protein-protein interactions, most of them present serious limitations. In a different study we have characterized a novel avian reovirus muNS-based protein tagging and inclusion targeting method, and demonstrated its validity to purify free an immobilized protein.

METHODOLOGY/PRINCIPAL FINDINGS:

Here we present a method to identify protein-protein interactions inside living eukaryotic cells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477-542), it not only got integrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to these structures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimeras that form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturing IC-tagged proteins, and that the proteins targeted to nuclear inclusions are able to interact with their known ligands.

CONCLUSIONS/SIGNIFICANCE:

Our protein redistribution method does not present the architectural requirement of re-constructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus of living cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration.

PMID:
21072177
PMCID:
PMC2970561
DOI:
10.1371/journal.pone.0013785
[Indexed for MEDLINE]
Free PMC Article

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