Etiquetado Intercelular de Librería sobre Librería para la Selección de Ligasa de Biotina y Péptidos Aceptores

Library-on-library (LOL) selection screens combinatorial libraries to generate new protein pairs. Previously, LOL selection has only been applied to stable protein-protein interactions. To extend LOL to transient enzyme-substrate pairs, a generalizable sequential LOL trans- and cis-labeling platform is developed, and a proof-of-concept selection is performed on Escherichia coli biotin ligase (BirA) and its acceptor peptide (AP). Using yeast surface display, AP mutant libraries are selected against BirA mutant libraries to identify AP variants trans-biotinylated by BirA mutants. Matched BirA mutants are subsequently enriched via the SpyTag-SpyCatcher-mediated cis-labeling platform. This represents the first demonstration of enzyme-peptide substrate LOL selection and offers a versatile framework for engineering new enzyme-peptide substrate pairs with varied activities.