Engineering the tryptophanyl tRNA synthetase and tRNATRP for the orthogonal expansion of the genetic code

textOver the last twenty years, the expansion of the genetic code has been made possible by the encoding of unnatural amino acids into proteins. Unnatural amino acids could be used to expand the chemical functionalities available to biology allowing for the production of ‘allo-proteins’ with potentially novel structures and functions. One method to engineer the genetic code is to engineer the translational components responsible for its maintenance. This methodology relies primarily on the evolution of the aminoacyl tRNA synthetases and their cognate tRNAs to produce an orthogonal enzyme and tRNA pair that allows for the insertion of unnatural amino acids into proteins. To date only a handful of these orthogonal pairs are available for use in genetic code expansion. As in vitro and in vivo techniques to re-code the genetic code have expanded, the utility of having multiple orthogonal pairs to site-specifically insert multiple unnatural amino acids into proteins has increased. In addition, the development of a variety of orthogonal pairs based on the twenty canonical aminoacyl tRNA synthetase-tRNA pairs will expand the types of unnatural amino acid sidechains available for protein engineering efforts. Herein we describe the engineering of the tryptophanyl tRNA synthetase and tRNA superscript Trp], pair from yeast for use as an orthogonal pair in E. coli. We have successfully built and tested synthetic expression constructs for the expression of this orthogonal pair in vivo. In addition, we have rationally engineered an orthogonal amber nonsense suppressor tRNA based on the yeast tRNA[superscript Trp], dubbed AS3.4. This suppressor has been shown to be an efficient orthogonal suppressor tRNA in vivo, and will aid in our efforts to expand the genetic code with heterocyclic unnatural amino acids. We also have developed a potentially tunable two part selection scheme, for use in the directed evolution of mutant tRNA synthetases that are specific to unnatural amino acid substrates.Biochemistr