Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System

Abstract

Constrained peptides are an attractive class as affinity reagents or drug leads owing to their excellent binding properties. Many kinds of these peptides, such as cyclic peptides containing disulfide bridges, are found in nature or designed artificially by directed evolution. However, confirming the binding properties of the disulfide-rich peptides can be generally difficult, because of oxidative folding problems in the preparation steps. Therefore, a method for evaluating the binding properties of such peptides rapidly and easily is required. Here, we report an easy and rapid method for preparing biotin-attached peptides containing disulfide bridges or a chemical cross-linker using a cell-free translation system and a puromycin-linker, which is applicable to pull-down assays for protein (or peptide) molecular interaction analysis.

Keywords

• molecular interaction analysis
• pull-down assay
• cell-free translation system
• puromycin
• constrained peptide
• cyclic peptide
• disulfide-rich peptide
• cross-linking
• in vitro selection
• directed evolution