A simple assay for screening translational activity of non-natural amino acids. Implications for polymer synthesis on messenger RNA templates

Abstract

The ability to incorporate unnatural amino acids into biologically synthesized proteins will greatly extend the impact of protein engineering on polymer materials science. The present report describes the use of a rapid cell-free assay to assess the incorporation potential of unnatural amino acids in Escherichia coli. The assay features a coupled transcription-translation system (‘Zubay system’) to screen incorporation of amino acid analogs into plasmid-encoded proteins. Activity estimates are based on the ability of an analog to compete with a radiolabeled natural amino acid, and toxicity effects are screened by monitoring incorporation of a second, unrelated amino acid. The assay was established with analogs known to be active in vivo, using a common bacterial expression vector as template DNA. Positive results were obtained with the leucine analog 5,5,5-trifluoroleucine, the proline analogs azetidine-2-carboxylic acid and thiazolidine-4-carboxylic acid, and three isomers of mono-fluorophenylalanine (o,m,p). No activity was observed for the phenylalanine analogs 2-thienylalanine and 3-thienylalanine. The results suggest that the cell-free assay will be a useful predictor of in vivo incorporation and a useful tool in the design and synthesis of genetically engineered materials.