title: Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli.
abstract: The site-specific incorporation of unnatural amino acids (UAAs) into proteins in living cells relies on an engineered tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair, orthogonal to the host cell, to deliver the UAA of choice in response to a unique nonsense or frameshift codon. Here we report the generation of mutually orthogonal prolyl-tRNA/prolyl-tRNA synthase (ProRS) pairs derived from an archaebacterial ancestor for use in Escherichia coli. By reprogramming the anticodon-binding pocket of Pyrococcus horikoshii ProRS (PhProRS), we were able to identify synthetase variants that recognize engineered Archaeoglobus fulgidus prolyl-tRNAs (Af-tRNA(Pro)) with three different anticodons: CUA, AGGG, and CUAG. Several of these evolved PhProRSs show specificity toward a particular anticodon variant of Af-tRNA(Pro), whereas others are promiscuous. Further evolution of the Af-tRNA(Pro) led to a variant exhibiting significantly improved amber suppression efficiency. Availability of a prolyl-tRNA/aaRS pair should enable site-specific incorporation of proline analogs and other N-modified UAAs into proteins in E. coli. The evolution of mutually orthogonal prolyl-tRNA/ProRS pairs demonstrates the plasticity of the tRNA-aaRS interface and should facilitate the incorporation of multiple, distinct UAAs into proteins.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Chatterjee
authors.forename: Han
authors.initials: H
authors.lastname: Xiao
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D019611:Archaeoglobus fulgidus / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli; D019143:Evolution, Molecular; D015723:Gene Library; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D043902:Pyrococcus horikoshii / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase