Dias, Ana Margarida Gonçalves CarvalhoTeixeira, Gonçalo Duarte GomesBarbosa, Arménio Jorge MouraGoncalves, JoaoIranzo, OlgaRoque, Ana Cecília Afonso2025-07-292025-07-292025-060961-8368PURE: 123128713PURE UUID: 3c67e920-a00b-45a8-8333-d66f52752ecdScopus: 105005970811PubMedCentral: PMC12095922ORCID: /0000-0002-9756-4958/work/188867194ORCID: /0000-0001-7416-7215/work/188867970http://hdl.handle.net/10362/185705Funding Information: The authors would like to acknowledge the financial support of FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB, projects UIDB/04138/2020 e UIDP/04138/2020 of the Research Institute for Medicines (iMed) and Gonçalo Teixeira PhD grant (PD/BD/139800/2018; COVID/BD/152648/2022). This work has received funding from the European Union's Horizon 2020 programme under grant agreement No. 899732 (PURE Project) and from Fundação para a Ciência e Tecnologia (Portugal) and ERDF under the PT2020 Partnership Agreement (LISBOA-01-0145-FEDER-028878) for funding the Sea2See project (PTDC/BII-BIO/28878/2017) and Proteios project (PTDC/CTM-CTM/3389/2021). Publisher Copyright: © 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.Protein engineering is a powerful tool in biotechnology and the basis to create unprecedented sequences, structures, and functions. The WW domains are a family of naturally occurring proteins involved in the molecular recognition of proline-rich and phosphorylated peptide sequences with relevance in cellular processes involved in human diseases. Due to their small size, WW domains represent appealing small protein domains for protein engineering and to generate novel functions as binders to non-cognate targets. Here, we designed a synthetic protein scaffold library based on the WW prototype sequence in which the loops were extended and randomized while maintaining structural stability. Using in vitro evolution by phage display against human serum albumin (HSA), we found a lead candidate that was produced by biological and chemical means and further characterized using experimental and computational tools. As a potential application for the lead binder, it was immobilized on a matrix and used to capture the target HSA. Overall, this work shows the versatility of WW domains as peptide scaffolds amenable for in vitro evolution against non-cognate targets.1630445384engAffinity purificationHuman serum albuminPhage displayProtein engineeringWW domainsBiochemistryMolecular BiologySDG 3 - Good Health and Well-beingjournal article10.1002/pro.70164https://www.scopus.com/pages/publications/105005970811https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=nova_api&SrcAuth=WosAPI&KeyUT=WOS:001492407100001&DestLinkType=FullRecord&DestApp=WOS_CPL