Rapid identification of functional pyrrolysyl-tRNA synthetases via fluorescence-activated cell sorting

The orthogonal pyrrolysyl-tRNA synthetase/tRNA _CUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically, herein we report a streamlined protocol for rapid construction of the pyrrolysyl-tRNA synthetase library, selection of the functional PylRS mutants using fluorescence-activated cell sorting, and subsequent validation of the selected PylRS mutants through direct expression of the fluorescent protein reporter using a single bacterial strain. We expect that this protocol should be generally applicable to rapid identification of the functional PylRS mutants for charging a wide range of non-natural amino acids into proteins.