UNIT 19.15 Identifying Small-Molecule Modulators of Protein-Protein Interactions
Published Online: 1 DEC 2006
Copyright © 2006 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Horswill, A. R. and Benkovic, S. J. 2006. Identifying Small-Molecule Modulators of Protein-Protein Interactions. Current Protocols in Protein Science. 46:19.15:19.15.1–19.15.19.
- Published Online: 1 DEC 2006
- Published Print: NOV 2006
This unit outlines methods for identifying cyclic peptides that inhibit protein-protein interactions. Proteins of interest are cloned into a two-hybrid system engineered to operate in reverse, allowing the disruption of a protein complex to be coupled to cell growth. Cyclic peptide libraries are generated using an intein-based plasmid construct, and the cyclized sequence is randomized using a PCR procedure. By transforming plasmid libraries into host cells containing the two-hybrid fusions, cyclic peptide inhibitors can be identified by growing the cells under the appropriate selective conditions. A detailed procedure for performing the genetic selection and identifying false positives is provided. Methods for building the two-hybrid protein fusions and optimizing media conditions, as well as an additional protocol for constructing cyclic peptide libraries are also provided.
- cyclic peptide;