Using the beta-lactamase protein-fragment complementation assay to probe dynamic protein-protein interactions.
We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. We have described a number of assays with different reporter readouts, but of particular value to studies of protein interaction dynamics are assays based on enzyme reporters that catalyze the creation of products, thus taking advantage of the amplification of signal afforded. Here we describe protocols for one such PCA based on the enzyme TEM beta-lactamase as a reporter in mammalian cells. The beta-lactamase PCA consists of fusing complementary fragments of beta-lactamase to two proteins of interest. If the proteins interact, the fragments are brought together and fold into active beta-lactamase. Here we describe a protocol for this PCA that can be completed in a few hours, using two different substrates that are converted to fluorescent or colored products by beta-lactamase.
Remy
Ingrid
I
Département de Biochimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada.
Ghaddar
Galia
G
Michnick
Stephen W
SW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003124:Colorimetry; D006801:Humans; D008856:Microscopy, Fluorescence; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases