Identifying small-molecule modulators of protein-protein interactions.
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.
Horswill
Alexander R
AR
University of Iowa, Iowa City, Iowa.
Benkovic
Stephen J
SJ
The Pennsylvania State University, University Park, Pennsylvania.
D001483:Base Sequence; D017931:DNA Primers; D005822:Genetic Vectors; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*
D017931:DNA Primers; D019151:Peptide Library; D010456:Peptides, Cyclic; D011506:Proteins