Final Report Summary - PROTLEGO (Development of an accessible platform for ex vivo site specific post-translational modifications of proteins)
The incorporation of unnatural amino acids (more than 100 to date) into proteins in vivo has resulted in the generation of proteins with novel chemical, biological and physical properties. However, a number of limitations restrict the incorporation of many unnatural amino acids into proteins in vivo: many unnatural amino acids do not cross the cell membrane; toxic unnatural amino acids cannot be incorporated in vivo since they damage the organism; and last, but not least, even if the unnatural amino acid does cross the cell membrane, the efficiency of transport is very low. We were able to overcome these limitations by exploiting translational components, evolved tRNA-synthetases and their cognate suppressor-tRNA from Archea, for the incorporation of an array of unnatural amino acids into proteins in a cell-free protein translation system. The advantage of the cell free system is that we were able to demonstrate our novel approach by incorporation of novel unnatural amino acids: into proteins using the broad substrate specificity of tRNA synthetases. We were the first to show that the pyrollysil translational components can be used in a cell free system for protein translation, thus paving the way for the incorporation of more than 50% of the available unnatural amino acids. In addition we could expand the genetic code for two different "words" rather than only one, which renders the system even more versatile and flexible for protein modification. The newly developed genetically recoded organism (GRO) E. coil was successfully utilised and adapted for our in house developed cell free transcription/translation system. Hence, we have developed a completely new platform and set of tools for the expansion of the genetic code in a cell free transcription/translation system with high fidelity and efficiency for the first time.