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For example, some two-hybrid systems use only one hybrid protein (e.g., G protein fusion systems), and with some PCAs, the refolded protein is not the final reporter by itself but initiates a process that results in the appearance of the actual reporter (e.g., split-ubiquitin system). These two definitions need to be taken with some practical flexibility. On the other hand, two-hybrid systems do not depend on PPI-induced refolding of protein fragments but rather on the colocalization of two protein domains ( Fig. The clear distinction between these groups lies in the fact that PCAs depend on the PPI-induced refolding of two protein fragments to reconstitute a functional reporter ( Fig. The technologies described can be divided into two main categories: two-hybrid systems and PCAs. In this review, we focus on genetic in vivo methods for PPI studies. Technologies in italics are not discussed in this review. ChIP-seq, chromatin immunoprecipitation followed by sequencing SELEX, systematic evolution by exponential enrichment PCA, protein fragment complementation assay. Overview of protein-protein interaction technologies and alternative applications for two-hybrid assay-derived methods. Affinity purification is the method of preference for the characterization of stable multiprotein complexes, in contrast to the yeast two-hybrid system, which is more suitable for identification of transient and binary PPIs. Different techniques are also complementary in the identity of PPIs that can be investigated. While biophysical methods such as isothermal titration calorimetry have the advantage of giving details on the kinetics of an interaction, several biochemical and genetic techniques can be used to screen for the identification of undiscovered binding partners. 1) is explained by the complementary output that each of them provides. The broad spectrum of available technologies ( Fig. Genetic approaches comprise phage display ( 78), the yeast two-hybrid system ( 178), protein fragment complementation assays (PCAs) ( 312), and protein microarrays ( 327). Examples of biochemical interaction technologies are the proximity ligation assay ( 606), cross-linking ( 661), the pulldown assay ( 67), coimmunoprecipitation ( 316), and tandem affinity purification (TAP) ( 524). Other biophysical methods include dual polarization interferometry ( 111), surface plasmon resonance ( 567), static light scattering ( 18), and circular dichroism ( 218) methods.
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These include isothermal titration calorimetry ( 514), where emission of heat during a protein association is analyzed, and fluorescence anisotropy ( 417), in which the reduced speed of rotational movement of a protein is detected after it binds another protein. Many genetic, biochemical, biophysical, and computational technologies are now developed that contribute to the knowledge on which proteins interact with each other, taking advantage of specific phenomena that occur during an interaction. The creativity and technical diversity that reside in the tools developed to investigate the binding of one protein to another show how eagerly scientists have been anticipating the characterization of protein-protein interactions (PPIs), from a small-scale atomic level to a large-scale interactomics level. Applications of these technologies are discussed, together with the advantages and limitations of the available assays. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli, but also in higher eukaryotes. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Summary: The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method.