Abstract
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.