A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries

Abstract

AbstractMany bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical peptides and target proteins fused to the GAL4-activating and binding domains, respectively. The library was constructed to contain triple-helical peptides with a “host-guest” design in which host helix-stabilizing regions flanked guest random sequences. Using this system, we selected peptides that bind to pigment epithelium-derived factor (PEDF), a collagen-binding protein that shows anti-angiogenic and neurotrophic activities, from the following libraries. Two-step selections from the total random library and subsequently from the second focused library yielded novel PEDF-binding sequences that exhibited affinity comparable to or more potent than that of the native PEDF-binding sequence in collagen. The sequences also contained a variant of the PEDF-binding motif that did not match the known motif identified from the native collagen sequences. This combinatorial library system allows the chemical space of triple-helical peptides to be screened more widely than that found in native collagen, thus increasing the expectation of obtaining more specific and high-affinity peptides.SignificanceCombinatorial random libraries of structurally constrained peptides have been constructed to obtain bioactive peptides with high affinity and/or biostability. This is the first report on the construction of a random collagen-like triple-helical peptide library by a DNA-encoded method and on an easy-to-access method of selecting triple-helical peptides that bind to proteins of interest. We identified several triple-helical peptides with unnatural sequences that bind to a collagen-binding protein. The binding affinity of these peptides was comparable to that of the peptide with the natural binding sequence in collagen. Our system demonstrates the feasibility of obtaining bioactive triple-helical peptides that could be used as drug leads or bioactive moieties of artificial extracellular matrices.