Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Abstract

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. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. 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. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli , but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. 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. Applications of these technologies are discussed, together with the advantages and limitations of the available assays.

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

“…A range of conceptually divergent approaches is currently available that differ in many aspects of the type of data they produce and the proteome subset they cover (8). In the quest for a more complete coverage of the interactome space, the value of such a diversified toolbox is underscored by the observation that different techniques detect different PPI subsets (17).…”

“…FRET and BRET, which rely on fluorescence or bioluminescence energy transfer between interacting fusion proteins, make assays with high spatiotemporal resolution (6,7). A variety of PCAs have been reported, including split fluorescent protein or reporter enzyme technologies, that are able to capture aspects of PPI dynamics in a mammalian background (8,9). A recent addition is an infrared fluorescent PCA that, unlike previous fluorescent PCAs, exhibits reversible complementation, thus enabling spatiotemporal analysis of dynamic PPIs (10).…”

Sensor–response regulator interactions in a cross-regulated signal transduction network

“…Interaction between T25 and T18 hybrid proteins generates cAMP, which activates lacZYA operon expression in an E. coli Dcya host strain lacking endogenous adenylate cyclase activity (Brickman et al, 1973). The BACTH method is particularly well-suited for analysing cytoplasmic membrane proteins (Karimova et al, 2005), because the two-hybrid interaction does not have to take place near the transcription complex as is required for most other interaction assays, including the yeast two-hybrid method (Stynen et al, 2012).…”

“…We set out to characterize intra-and intermolecular interactions between these cognate pairs, in order to gain insights into specificity and cross-regulatory determinants. For this purpose, we employed the BACTH method (bacterial adenylate cyclase two-hybrid) (Karimova et al, 1998), a widely used genetic approach for monitoring protein-protein interactions (Stynen et al, 2012). This assay reconstitutes Bordetella pertussis CyaA activity from trypsin-defined amino-terminal and carboxyl-terminal subdomains of 25 and 18 kDa, respectively (Guo et al, 2005).…”

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

“…To be able to quantify the rate and efficiency of delivery we used a split fluorescent protein (Venus) which is only one of a rapidly growing list of proteins that can be split and then functionally complemented. Therefore, this approach could easily be extended to a large number of proteins through either trial and error or based on structural information and should be applicable to single globular domains as well as multidomain proteins [45,46]. …”