A SIMPLE ASSAY FOR SCREENING TRANSLATIONAL ACTIVITY OF NONNATURAL AMINO-ACIDS - IMPLICATIONS FOR POLYMER SYNTHESIS ON MESSENGER-RNA TEMPLATES

The ability to incorporate unnatural amino acids into biologically syn thesized proteins will greatly extend the impact of protein engineerin g on polymer materials science. The present report describes the use o f a rapid cell-free assay to assess the incorporation potential of unn atural amino acids in Escherichia coil. The assay features a coupled t ranscription-translation system ('Zubay system') to screen incorporati on of amino acid analogs into plasmid-encoded proteins. Activity estim ates are based on the ability of an analog to compete with a radiolabe led natural amino acid, and toxicity effects are screened by monitorin g incorporation of a second, unrelated amino acid. The assay was estab lished with analogs known to be active in vivo, using a common bacteri al expression vector as template DNA. Positive results were obtained w ith the leucine analog 5,5,5-trifluoroleucine, the proline analogs aze tidine-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 predicto r of in. vivo incorporation and a useful tool in the design and synthe sis of genetically engineered materials. (C) 1995 John Wiley and Sons, Inc.