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Reverse Yeast Two-hybrid (rY2H) Service

Creative Biolabs can offer reverse yeast two-hybrid (rY2H) system services for detection of protein-protein interactions. As a genetic method, rY2H has been widely used in drug screening.

Description of rY2H

Protein-protein interactions take part in most biological processes of cells including gene expression, cell growth, proliferation, morphology, motility, intercellular communication. And the use of yeast two-hybrid (Y2H) system has provided definition to previously unknown pathways by the characterization of novel interactions between proteins. The two-hybrid system relies on the bifunctional nature of transcription factors, such as yeast enhancer Gal4 or repressor LexA to allow protein-protein interactions to be monitored through transcriptional changes of reporter genes. When a positive interaction has been validated, either of the interacting proteins can be mutated by site-specific or randomly introduced changes, to generate proteins with a decreased capability to interact. Such technique is termed reverse two-hybrid (rY2H/rYTH) method, specifically designed to promote identification of events isolating protein-protein interactions. More attention will shift to this powerful search engine for the establishment of interaction networks as well as drug discovery in understanding of diseases.

Reverse Yeast Two-hybrid (rY2H) Service Fig.1 Reverse yeast two-hybrid (rY2H) for drug screening.

The reverse two-hybrid system is a genetic scheme that has far been described to discern molecules or mutations that dissociate specific interacting partners for a protein of interest. In Saccharomyces cerevisiae, yeast strains are engineered so that the interaction of two proteins expressed in the context of the counterselective system is deleterious to growth. Under these conditions, dissociating interactome provides an alternative survival advantage, thereby facilitating detection. This method contributes to the study of the structure-function relationships and regulation of protein-protein interactions. It also should accelerate the selection of dissociator which can be used as therapeutic agents.

Mechanisms of rY2H

Conceptually, rY2H makes use of a contrary strategy to produce a positive readout for disruption of the designated interactions of proteins. Hence, no parallel toxicity testing of the complexes is required in the screening procedure. That is to say, in this upside-down version of Y2H, the wild-type protein interaction is toxic or lethal for the yeast cells because of a toxic marker (e.g. URA3). In this setting, dissociation confers a selective growth advantage that can easily identify both interacting proteins and trans-acting dissociators or small molecules. The lynchpin of rY2H is the incorporation of a reporter gene, to monitor protein-protein interactions, whose final products are poisonous to the living cells. This allows the utilization of selective pressure against the formation of two-hybrid compounds.

Reverse Yeast Two-hybrid (rY2H) Service Fig.2 An illustration of the way two-hybrid systems work.

The yeast gene URA3 encodes an enzyme involved in uracil biosynthesis and transforms 5-flouroorotic acid (5-FOA) into a deleterious metabolite that leads to cell death. Therefore, yeast cells that express URA3 fail to grow on media containing 5-FOA and gene URA3 is commonly chosen as a counter-selectable marker (negative selection). There is a yeast strain engineered in which expression of URA3 is controlled by a strictly regulated promoter containing GAL4 binding sites. Expression of interacting GAL4 activation domain (AD) with GAL4 DNA-binding domain (DBD) rescues the viability of this strain on media lacking uracil, while growth on complete media adding 5-FOA is inhibited by interacting AD and DBD fusions. As a result, dissociating mutations in interacting proteins can be isolated from a large library of randomly generated mutants by selection for 5-FOA-resistant colonies.

Proteins are basic building components of cells and protein interactions and define key biochemical and regulatory networks. Protein-protein interactions are an essential aspect of all signaling pathways and biological mechanisms and are strongly predictive of functional relationships.

Reverse Yeast Two-hybrid (rY2H) Service Fig.3 Forward and reverse n-hybrid system.

Theoretically, in all diseases attributed to particular protein-protein interactions, special dissociation can be regarded as a potential therapeutic strategy. Identification of dissociator molecules could be achieved by direct selection through the reverse two-hybrid system. Creative Biolabs has experienced and advanced platforms in rY2H methods that are applied for abrogating defined macromolecular interactions and rational drug-screening. Besides that, Creative Biolabs can still offer other forward or reverse “n”-hybrid systems professionally to evaluate the complex interplay of proteins with DNAs, RNAs, peptide ligands, small organic ligands, protein kinases, etc.




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