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Mapping Biochemical Networks with Protein Fragment Complementation Assays

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Protein-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1278))

Abstract

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on Protein fragment Complementation Assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, etc.). In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and β-lactamase.

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Authors and Affiliations

  1. Département de Biochimie, Université de Montréal, C.P. 6128, succursale centre-ville, Montréal, QC, Canada, H3C 3J7

    Ingrid Remy & Stephen W. Michnick

Authors
  1. Ingrid Remy
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  2. Stephen W. Michnick
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Corresponding author

Correspondence to Stephen W. Michnick .

Editor information

Editors and Affiliations

  1. Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA

    Cheryl L. Meyerkord

  2. Department of Pharmacology, Department of Hematology & Medical Oncology, and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, USA

    Haian Fu

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Remy, I., Michnick, S.W. (2015). Mapping Biochemical Networks with Protein Fragment Complementation Assays. In: Meyerkord, C., Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 1278. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2425-7_31

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  • DOI: https://doi.org/10.1007/978-1-4939-2425-7_31

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2424-0

  • Online ISBN: 978-1-4939-2425-7

  • eBook Packages: Springer Protocols

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