Utilizing elongation factor Tu mutagenesis to incorporate large unnatural amino acids in vivo

Abstract

Site‐specific in vivo incorporation of unnatural amino acids (UAAs) via aminoacyl tRNA synthetase (RS) mutagenesis has been widely used in protein structure and function studies. While a large, diverse family of UAAs has been incorporated into proteins, aminoacyl tRNA synthetases for certain UAAs seem problematic to evolve or generate. We developed a reporter system to explore substrate specificity of current UAA RSs. Using these permissive RSs, structural limitations have been explored. The photocrosslinking UAAs anthraquinonylalanine and 3‐(3’‐fluorenyl‐9’‐oxo)‐alanine should be incorporated by permissive RSs. However, incorporation of these UAAs into proteins has not yet been accomplished. It is the current understanding that elongation factor Tu (EF‐Tu) size limitation is preventing the incorporation of these UAAs. It is believed that UAAs that were previously too large to see incorporation with mutant RSs alone will hopefully be incorporated utilizing mutant RS and mutant EF‐Tu combinations in vivo. Here we report progress with mutant EF‐Tu and orthogonal aminoacyl tRNA synthetase pairs. Supported by F&M funds, NSF‐MCB‐0448297; Research Corporation (CC6364).