title: Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells.
abstract: The genetic code of living organisms has been expanded to allow the site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon UAG. Numerous amino acids have been incorporated including photo-crosslinkers, chemical handles, heavy atoms and post-translational modifications, and this has created new methods for studying biology and developing protein therapeutics and other biotechnological applications. Here we describe a protocol for reprogramming the amino-acid substrate specificity of aminoacyl-tRNA synthetase enzymes that are orthogonal in eukaryotic cells. The resulting aminoacyl-tRNA synthetases aminoacylate an amber suppressor tRNA with a desired unnatural amino acid, but no natural amino acids, in eukaryotic cells. To achieve this change of enzyme specificity, a library of orthogonal aminoacyl-tRNA synthetase is generated and genetic selections are performed on the library in Saccharomyces cerevisiae. The entire protocol, including characterization of the evolved aminoacyl-tRNA synthetase in S. cerevisiae, can be completed in approximately 1 month.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742-2021, USA. acropp@umd.edu
authors.forename: T Ashton
authors.initials: TA
authors.lastname: Cropp
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Anderson
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D005057:Eukaryotic Cells; D015723:Gene Library; D005815:Genetic Code; D008781:Methyltyrosines / Q000737:chemistry / Q000235:genetics; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D014170:Transformation, Genetic
chemical_list: D000596:Amino Acids; D008781:Methyltyrosines; C002350:O-methyltyrosine; D000604:Amino Acyl-tRNA Synthetases