Embryonic Stem Cells

Volume 185 of the series Methods in Molecular Biology™ pp 447-459

Detection and Visualization of Protein Interactions with Protein Fragment Complementation Assays

  • Ingrid RemyAffiliated withDepartment of Biochemistry, University of Montréal
  • , André GalarneauAffiliated withDepartment of Biochemistry, University of Montréal
  • , Stephen W. MichnickAffiliated withDepartment of Biochemistry, University of Montréal

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A first step in defining the function of a novel gene is to determine its interactions with other gene products in an appropriate context; that is, because proteins make specific interactions with other proteins as part of functional assemblies, an appropriate way to examine the function of the product of a novel gene is to determine its physical relationships with the products of other genes. This is the basis of the highly successful yeast two-hybrid system (16). The central problem with two-hybrid screening is that detection of protein-protein interactions occurs in a fixed context, the nucleus of S. cerevisiae, and the results of a screening must be validated as biologically relevant using other assays in appropriate cell, tissue or organism models. While this would be true for any screening strategy, it would be advantageous if one could combine library screening with tests for biological relevance into a single strategy, thus tentatively validating a detected protein as biologically relevant and eliminating false-positive interactions immediately. It was with these challenges in mind that our laboratory developed the protein-fragment complementation assay (PCA) strategy. In this strategy, the gene for an enzyme is rationally dissected into two pieces. Fusion proteins are constructed with two proteins that are thought to bind to each other, fused to either of the two probe fragments. Folding of the probe protein from its fragments is catalyzed by the binding of the test proteins to each other and is detected as reconstitution of enzyme activity.