Application of Random Peptide Phage Display to the Study of Nuclear Hormone Receptors

Abstract

Listen

Translate article

This chapter focuses on the application of random peptide phage display to the study of nuclear hormone receptors. The chapter is organized into four sections: (1) the construction of a peptide phage display library, (2) a step-by-step protocol for biopanning (the affinity selection process), (3) the discussion of a mammalian cell-based screening technology to validate and complement the in vitro phage display approach, and (4) the use of phage display selected peptides as antagonists of nuclear receptor function. The selection of peptides that bind to the target proteins of interest is achieved by incubating phage libraries containing small random peptide inserts with the target proteins that are immobilized on a solid support. Phage display has been widely used as a method to map protein–protein interactions, identify peptide ligands for cell surface receptors, and map antibody–antigen epitopes. By using phage display, random peptide libraries can be generated and specific target-interacting peptides can be identified. The random peptide libraries are created by inserting short random oligonucleotides within the coding sequence of the M13 bacteriophage capsid protein, with the subsequent display of random peptides on the outer surface of the phage. In addition to using phage display to study receptor conformation and SERM pharmacology, this powerful technique has been used to develop highly specific peptide antagonists that block receptor function by interfering with required protein–protein interactions. A very good alternative to immobilizing steroid–nuclear hormone receptors and other DNA-binding transcription factors is by using biotinylated oligonucleotides (corresponding to their cognate response element) pre-captured on streptavidin-coated plates.