A yeast three-hybrid method to clone ternary protein complex components.
The yeast two-hybrid system is a powerful technique to detect binary protein interactions. In order to facilitate ternary complex analysis, we have developed the yeast three-hybrid system. Using epidermal growth factor (EGF) receptor, Grb2 and Sos proteins as an example, we demonstrated the use of the three-hybrid system to detect formation of a three-component complex, i.e., the Grb2-mediated interactions between EGF receptor and Sos. We then used the three-hybrid system to screen a library with Gal4-DNA binding domain -EGF receptor and Gal4 activation domain-Sos fusion proteins and isolated a plasmid encoding Grb2. Thus, the three-hybrid system is useful for characterizing and cloning components of ternary protein complexes.
Zhang
J
J
Guilford Pharmaceuticals, Inc., Baltimore, Maryland 21224, USA.
Lautar
S
S
D048868:Adaptor Proteins, Signal Transducing; D003001:Cloning, Molecular / Q000379:methods*; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D005800:Genes, Fungal; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D010957:Plasmids; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020664:Son of Sevenless Proteins; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D005656:Fungal Proteins; D051380:GRB2 Adaptor Protein; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020664:Son of Sevenless Proteins; D066246:ErbB Receptors