Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions.
The detection of physical interaction between two or more molecules of interest can be facilitated if the act of association between the interactive partners leads to the production of a readily observed biological or physical readout. Many interacting molecule pairs (X, Y) can be made to induce such a readout if X and Y are each fused to defined protein elements with desired properties. For example, in the yeast forward two-hybrid system, X is synthesized as a translational fusion to a DNA-binding domain (DBD), Y is synthesized as a fusion to a transcriptional activation domain (AD), and coexpression of DBD-X and AD-Y induces transcription of easily scored responsive reporters. Other approaches use paradigms based on the artificial production of two, hybrid, molecules, but substitute a variety of readouts including the repression of transcription, activation of signal transduction pathways, or reconstitution of a disrupted enzymatic activity. In this article, we summarize a number of two-hybrid-based approaches, and detail the use of the forward yeast two-hybrid system in a screen to identify novel interacting partners for a protein of interest.
Toby
G G
GG
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Golemis
E A
EA
D004274:DNA, Recombinant / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors / Q000378:metabolism; D066298:In Vitro Techniques; D019151:Peptide Library; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology; D014170:Transformation, Genetic / Q000502:physiology; D020798:Two-Hybrid System Techniques
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D019151:Peptide Library; D011506:Proteins; D012097:Repressor Proteins