The incorporation of unnatural amino acids (more than 50 to date) into proteins in vivo has resulted in the generation ofproteins with novel chemical, biological, and physical properties. However, some unnatural amino acids possess properties,such as an inability to cross the cell membrane or a level of toxicity dangerous to the organism, that restrict their incorporationinto proteins in vivo. In addition, even when an unnatural amino acid crosses the cell membrane, its transport efficiencywithin the cell is very low. We propose to overcome these limitations by exploiting translational components evolvedtRNA-synthetases and their cognate suppressor-tRNA from Archea for the incorporation of an array of unnatural amino acidsinto proteins in vitro in a cell-free protein translation system. The expressed recombinant proteins containing the unnaturalamino acids will be purified from the reaction mixture and used for further research. Using the cell free system, first we willdemonstrate our new approach by incorporating novel unnatural amino acids, i.e., thiolysine analogues, into proteins usingthe broad substrate specificity of evolved tRNA synthetases. We will then incorporate a thiolysine analogue into PCNA forthe site-specific ubiquitination and SUMOylation of these proteins for in vitro studies of the interactions between PCNA andinteracting proteins and to follow the progress of the replication fork. This unique approach will show for the first time the useof evolved synthetases in a cell free translation system, with the advantage being that previously un-incorporable unnaturalamino acids can be incorporated using this approach. Our overall aim is to enable the introduction of new functionalities intoproteins.