Incorporation of 3-hydroxyphenylalanine into proteins using aaRS/tRNA pair

Incorporation of non-canonical amino acids (ncAAs) into proteins has emerged as a powerful approach to modify the properties of natural proteins, leading to enhanced and expanded protein functions. Aromatic ncAAs represent a significant class of ncAAs that have been employed for engineering enzyme catalytic efficiency, protein structure studies, and the development of biomaterials. This study focused on the incorporation of 3-hydroxyphenylalanine (3HLP), a non-proteinogenic aromatic amino acid, and aims to establish a 3HLP incorporation system. We constructed an incorporation detection system based on the sfGFP fluorescent protein and screened out the chPheRS2/tRNACUA pair for 3HLP incorporation, expanding the application of aromatic ncAAs in protein engineering. In addition, molecular docking was analyzed by computer simulation.