In an effort to develop a method for the high-throughput analysis of protein interaction interfaces, we devised a novel yeast genetic screening method, termed the "one- plus two-hybrid system," which efficiently selects specific missense mutations that disrupt known protein-protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid screening system is used first to positively select intact prey proteins, harboring informative missense mutations, from a large library of randomly generated mutant alleles. Next, among the isolated missense mutants of the prey proteins, interaction-defective mutants for a given protein (bait) are selected using the two-hybrid screening system. As a validation of the feasibility of this method, we utilized this technique to rapidly characterize the molecular determinants of the interactions between vitamin D receptor and its transcriptional coactivator protein, thyroid hormone receptor-associated protein 220. This efficient and rapid method should prove useful in the systematic analysis of large numbers of interaction interfaces. © 2012 Springer Science+Business Media, LLC.
CITATION STYLE
Kim, J. Y., Park, O. G., & Lee, Y. C. (2012). One- plus two-hybrid system for the efficient selection of missense mutant alleles defective in protein-protein interactions. Methods in Molecular Biology, 812, 209–223. https://doi.org/10.1007/978-1-61779-455-1_12
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