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

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.

Residue-specific incorporation of unnatural amino acids into proteins in vitro and in vivo.

The incorporation of noncanonical (unnatural) amino acids into proteins offers researchers the ability to augment the biochemical functionality of proteins for a myriad of applications including bioorthogonal conjugation, biophysical and structural studies, and the enhancement or de novo creation of novel enzymatic activities. The augmentation of a protein throughout its coding sequence by global residue-specific incorporation of unnatural amino acid analogs is an attractive technique for studying both the utility of individual chemistries available through unnatural amino acids and the general effects of unnatural amino acid substitution on protein structure and function. Herein we describe protocols to introduce unnatural amino acids into proteins using the Escherichia coli translation system either in vivo or in vitro. Special attention is paid to obtaining high levels of incorporation while maintaining high yields of protein expression.