A two-step two-hybrid system to identify functionally significant protein-protein interactions.
The two-step two-hybrid approach described here is an adaptation of the classic two-hybrid system. Its purpose is to identify proteins that interact with a relatively small, defined, functionally significant domain of a protein of interest. In this method, a first round of screening is performed to identify proteins that interact with bait comprised of the wild type protein. Next, each of the prey identified in this first round is tested for its ability to interact with functionally impaired, mutant bait. Any proteins that interact with the wild type bait, but not the mutant bait, are candidate effectors or regulators of the protein of interest.
Poustovoitov
Maxim
M
Fox Chase Cancer Center, 333 Cotman Ave, Philadelphia, PA 19111, USA.
Serebriiskii
Ilya
I
Adams
Peter D
PD
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D018076:DNA, Complementary; D007930:Leucine; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D001616:beta-Galactosidase; D000099160:LexA Repressor Protein