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bioRxiv · 10.1101/2023.11.14.567114

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

Masuda, R.·Thant, K. P. P.·Kawahara, K.·Oki, H.·Kadonosono, T.·Kobayashi, Y.·Koide, T.
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Abstract

Many 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 having 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 libraries. Two-step selections from the total random library and subsequently from the second focused library yielded new PEDF-binding sequences that exhibited a comparable affinity to or more potent than that of the native PEDF-binding sequence in collagen. The obtained 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.

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Masuda, R., Thant, K. P. P., Kawahara, K., Oki, H., Kadonosono, T., Kobayashi, Y., Koide, T.. 2023-11-16. A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries. https://doi.org/10.1101/2023.11.14.567114

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