The Two Hybrid Dual Bait System: An Improved Two Hybrid System for the Detection of Protein-Protein Interactions
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To understand and manipulate the function of a particular protein of biological interest, it is generally useful to identify other proteins with which it associates. Identification of protein interactions initially proceeded by technically difficult biochemical methods. Over the last five years, however, yeast two-hybrid systems have emerged as a powerful tool for the identification of protein-protein interactions, and have been extensively used to isolate novel partners for proteins of interest. With continuing reagent development, two-hybrid systems have more recently been used for additional applications, including detection of RNA-binding proteins, identification of proteins for ligands and ligands for proteins, and identification of bioactive peptide sequences. At present, a major focus of research efforts has been to adapt two hybrid approaches to genomic analyses with the ultimate goal of developing interaction maps of protein-protein contacts.
Fox Chase researchers have created reagents that will greatly enhance the reliability and general utility of two hybrid Interaction Trap systems. The improved reagents incorporate controls for false positives or non-specific interactions in a single step and allow the simultaneous assay of a protein interaction with two related or non-related partners in a single cell.
The yeast two hybrid system has been enormously effective when applied to a great number of classes of proteins; a casual survey of Medline for the last four years identifies over 700 reports of papers utilizing two hybrid technologies to analyze protein function, with over 250 reports of identification of biologically significant interacting proteins from libraries.
Although these efforts have indicated that two hybrid systems generally work remarkably well, there are some particular instances where they are subject to error, supporting the idea that further improvements of reagents would be useful.
Development of the advanced Interaction Trap-based "Dual Bait System" is expected to produce several important benefits. First, it will allow library screens to be performed with an immediate selection to eliminate false positives. This will be extremely critical in some applications, such as screening for RNA-binding proteins, which have notably high backgrounds. Second, the improved system will allow comparative simultaneous assessment of interactions between an activation-domain-fused protein and two partner proteins which might be related members of a protein family, or a wild type versus mutated form of the same protein. This latter application is likely to be of particular use in conjunction with targeted drug discovery efforts.
The improved two hybrid bait system is available for licensing for in-house research.