UNIT 20.8 Interaction Trap/Two-Hybrid System to Identify Loss-of-Interaction Mutant Proteins
Published Online: 1 FEB 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Mendelsohn, A. R. 2004. Interaction Trap/Two-Hybrid System to Identify Loss-of-Interaction Mutant Proteins. Current Protocols in Molecular Biology. 65:20.8:20.8.1–20.8.10.
- Published Online: 1 FEB 2004
- Published Print: JAN 2004
Protein-protein interactions play a critical role in biology. Disrupting a specific protein-protein interaction and studying the resulting phenotype can help elucidate the function of the interaction. A method to rapidly make and identify mutant proteins that no longer bind to a specific partner protein is described. The method takes advantage of the ease of the interaction trap/two-hybrid system and PCR mutagenesis. PCR fusion of the target protein to GFP is used to ensure the protein's open reading frame is not disrupted by mutagenesis. The resulting noninteracting mutant proteins usually result from a single missense mutation, which can easily be identified.