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.
|Original language||English (US)|
|Pages (from-to)||Unit 19.15|
|Journal||Current protocols in protein science / editorial board, John E. Coligan ... [et al.]|
|State||Published - Dec 2006|
All Science Journal Classification (ASJC) codes
- Structural Biology