Unnatural Amino Acids (UAAs), amino acids not present in the human genetic code, have been synthesized to have a broad range of useful properties, particularly as probes and sensors which can be integrated directly into a protein. In order for the cell to place a UAA into the protein, two components, an aminoacyl tRNA synthetase and a corresponding tRNA developed, in this case, for the TAG (amber) codon must be present. If an amino acid is successfully charged to the tRNA, the amber codon in suppressed and a functional protein is built with the UAA at the mutation site. Such a tRNA molecule has previously been developed, as well as many synthetases specific to UAAs. In this work, the range of UAAs which can be incorporated into proteins using the <i>E. coli’s</i> own machinery is expanded by the development of novel aminoacyl tyrosine tRNA synthetases originating from <i>Methanococcus jannaschii</i> . By making a library of synthetase-coding plasmid variants and performing positive and negative screenings using the amber suppression method, the binding pocket of the synthetase can be modified for specificity to a UAA while not allowing the tRNA to be charged with a natural amino acid. In this work, we are attempting to evolve new tRNA synthetases that can incorporate either 3-(2-Pyridyl)-L-Alanine or L-Penicillamine unnatural amino acids into a protein in response to an amber codon using a nonsense mutated ampicillin resistance gene for the positive screen and a nonsense mutated barnase gene for the negative screen.  These two unnatural amino acids do not have any evolved synthetases currently available, and it would be beneficial to incorporate these into proteins due to their promising metal-binding capabilities.

Development of an unnatural amino acid-specific aminoacyl tRNA synthetase

255th National Meeting (2018)

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