Abstract
The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins via amber suppression provides access to novel protein properties, structures, and functions. Historically, poor protein expression yields resulting from release factor 1 (RF1) competition has limited this technology. To address this limitation, we develop a high-yield, one-pot cell-free platform for synthesizing proteins bearing ncAAs based on genomically recoded Escherichia coli lacking RF1. A key feature of this platform is the independence on the addition of purified T7 DNA-directed RNA polymerase (T7RNAP) to catalyze transcription. Extracts derived from our final strain demonstrate high productivity, synthesizing 2.67 ± 0.06 g/L superfolder GFP in batch mode without supplementation of purified T7RNAP. Using an optimized one-pot platform, we demonstrate multi-site incorporation of the ncAA p-acetyl-L-phenylalanine into an elastin-like polypeptide with high accuracy of incorporation and yield. Our work has implications for chemical and synthetic biology.