Proteins typically use specific arrangements of the natural twenty amino acids. Expanding the set of amino acids can both aid in the study of these macromolecules as well as significantly increase their bioavailability and robustness. A set of enzymes known as aminoacyl-tRNA synthetases lie at the heart of the fidelity of translation. These synthetases accurately charge a specific tRNA with its cognate amino acid in spite of the presence of other amino acids. However, systems have been developed in which a novel suppressor tRNA and an orthogonal aminoacyl-tRNA synthetase incorporate unnatural amino acids (unAA) with properties outside of the natural set. Our aim was to adapt this strategy to produce complex proteins, containing unnatural amino acids, within a cell-free protein synthesis system.

The openness of the cell-free system allows precise control of both natural and unAA concentrations, avoiding cellular uptake limitations, as well as the concentrations of the orthogonal suppressor tRNA and aminoacyl-tRNA synthetase. In addition it significantly reduces the period for protein expression and folding (3 to 6 hours) as compared to two weeks for mammalian cell processes. It is also advantageous to use cell-free protein technology because it can be used to produce toxic proteins and offers high product quality, low production cost, and low capital costs.

We have initially chosen to incorporate O-methyl-L-tyrosine, p-acetyl-L-phenylalanine, and p-azido-L-phenylalanine site-specifically using an orthogonal tRNATyr/Tyrosine-synthetase pair from Methanococcus jannaschii. We have incorporated these unnatural amino acids into soluble bacterial proteins; therapeutic, mammalian secreted proteins containing disulfide bonds, and membrane proteins. This cell-free protein synthesis platform was developed by optimizing cell strains, extract preparation protocols, chaperone concentrations, and cell-free reaction conditions. Reactions in which the tRNA and synthetase are either purified and added to the cell-free reaction or produced during cell extract preparation result in significant quantities of disulfide bonded and vesicle incorporated proteins. Activity was verified by colorimetric, cell proliferation, immunoprecipitation, and transport assays. Improved incorporation of unAA's has motivated us to attempt targeted post-translational modification of the azide and ketone moieties inserted into the cell-free products. This may allow us to rapidly produce functionalized membrane proteins and homogeneous therapeutics and vaccines.