Protein interactions: analysis using allele libraries.
Interaction defective alleles (IDAs) are alleles that contain mutations affecting their ability to interact with their wild type binding partners. The locations of the mutations may lead to the identification of protein interaction domains and interaction interfaces. IDAs may also distinguish different binding interfaces of multidomain proteins that are part of large complexes, thus shedding light on large protein structures that have yet to be determined. IDAs may also be used in conjunction with RNAi to dissect protein interaction networks. Here, the wild type allele is knocked down and replaced with an IDA that has lost the ability to interact with a specific binding partner. As a result, interactions are disrupted rather than knocking out the entire gene. Thus, IDAs have the potential to be extremely valuable tools in protein interaction network analysis. IDAs can be isolated by reverse two-hybrid analysis, which was demonstrated over a decade ago, but high background levels caused by truncated IDAs have prevented its widespread adoption. We recently described a novel method for full-length allele library generation that eliminates this background and increases the efficiency of the reverse two-hybrid protocol (and IDA isolation) significantly. Here we discuss our strategy for allele library generation, the potential uses of IDAs as outlined above, and additional applications of allele libraries.
Vista Biologicals, 2120 Las Palmas Drive, 92011, Carlsbad, CA, USA.
Thomas G
TG
Chappell
Phillip N
PN
Gray
D000483:Alleles; D019943:Amino Acid Substitution; D015723:Gene Library; D009154:Mutation; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D011506:Proteins