High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNACUAopt and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.

Graphical abstractCell-free synthesis.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Cell-free synthesis affords high protein yields with unnatural amino acids. ► 4-(Trifluoromethyl)phenylalanine incorporated by a polyspecific synthetase. ► Truncation at amber stop codons can be suppressed almost completely. ► Two unnatural amino acids can readily be incorporated at two different sites.