An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins

• We have developed a CFPS system for global unnatural amino acid incorporation into proteins.
• This new system is efficient, high-yield, low background, cost-effective and easy to produce.
• We compare our system to commercially available alternatives, and traditionally made CFPS systems.
• Functional and physical data for two target proteins demonstrate our system's high-yield and extremely low background.
• We show that streptavidin is functional even when the tryptophan residues are replaced with fluorinated tryptophans.

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.