One- plus two-hybrid system for the efficient selection of missense mutant alleles defective in protein-protein interactions.
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.
School of Biological Sciences and Technology, Chonnam National University, Gwangju, Republic of Korea.
Ji Young
JY
Kim
Ok Gu
OG
Park
Young Chul
YC
Lee
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D020125:Mutation, Missense / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D011506:Proteins