Membrane-based yeast two-hybrid system to detect protein interactions.
The classical yeast two-hybrid system and its modifications have been successfully used over the past decade to investigate interactions between most classes of proteins expressed in a given cell or tissue. However, some proteins (e.g., integral membrane proteins or nuclear proteins) are relatively difficult to investigate by standard yeast two-hybrid methods either because they are retained at cellular membranes or they activate the system in the absence of a true protein interaction. The membrane-based yeast two-hybrid (MbY2H) system presented in this unit overcomes some of these limitations. It is based on the split-ubiquitin protein complementation assay and detects protein interactions directly at the membrane, thereby allowing the use of full-length integral membrane proteins and membrane-associated proteins as baits to hunt for novel interaction partners. A simple modification also allows the use of proteins that are self-activating in a classical yeast two-hybrid system (e.g., acidic proteins and many transcription factors). Like the yeast two-hybrid system, the MbY2H system can also be used for interaction discovery by screening complex cDNA libraries for novel interaction partners.
Lentze
Nicolas
N
Dualsystems Biotech, Schlieren, Switzerland.
Auerbach
Daniel
D
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D015723:Gene Library; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D011506:Proteins