Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins

• Published in: Scientific reports Vol. 13; no. 1; p. 1339
• Main Authors: Gomes, M., Fleck, A., Degaugue, A., Gourmelon, F., Léger, C., Aumont-Nicaise, M., Mesneau, A., Jean-Jacques, H., Hassaine, G., Urvoas, A., Minard, P., Valerio-Lepiniec, M.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 24-01-2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/45/612; 631/61/338; Amino Acid Sequence; Bacteriophages - genetics; Binders; Cell Surface Display Techniques; CheY protein; Humanities and Social Sciences; multidisciplinary; Nucleotide sequence; Peptide Library; Phage display; Proteins; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-27710-4

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from
Thermotoga maritima
was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the
Oplophorus
luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the μM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.