LAUSR

Incorporation of noncanonical amino acids (ncAAs) into proteins opens new opportunities in biotechnology and synthetic biology. Pyrrolysine (Pyl)‐based ncAAs are some of the most predominantly used, but expression systems suffer from low yields. Here, we report a highly efficient cell‐free protein synthesis (CFPS) platform for site‐specific incorporation of Pyl‐based ncAAs into proteins using amber suppression. This platform is based on cellular extracts derived from genomically recoded Escherichia coli lacking release factor 1 and enhanced through deletion of endonuclease A. To enable ncAA incorporation, orthogonal translation system (OTS) components (i.e., the orthogonal transfer RNA [tRNA] and orthogonal aminoacyl tRNA synthetase) were coexpressed in the source strain prior to lysis and the orthogonal tRNACUAPyl that decodes the amber codon was further enriched in the CFPS reaction via co‐synthesis with the product. Using this platform, we demonstrate production of up to 442 ± 23 µg/mL modified superfolder green fluorescent protein (sfGFP) containing a single Pyl‐based ncAA at high (>95%) suppression efficiency, as well as sfGFP variants harboring multiple, identical ncAAs. Our CFPS platform can be used for the synthesis of modified proteins containing multiple precisely positioned, genetically encoded Pyl‐based ncAAs. We anticipate that it will facilitate more general use of CFPS in synthetic biology.