title: Traceless and site-specific ubiquitination of recombinant proteins.
abstract: Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of δ-thiol-L-lysine (7) and δ-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
authors.forename: Satpal
authors.initials: S
authors.lastname: Virdee
authors.forename: Prashant B
authors.initials: PB
authors.lastname: Kapadnis
authors.forename: Thomas
authors.initials: T
authors.lastname: Elliott
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lang
authors.forename: Julia
authors.initials: J
authors.lastname: Madrzak
authors.forename: Duy P
authors.initials: DP
authors.lastname: Nguyen
authors.forename: Lutz
authors.initials: L
authors.lastname: Riechmann
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001665:Binding Sites; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D017021:Methanosarcina barkeri / Q000201:enzymology; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D054875:Ubiquitination
chemical_list: D011994:Recombinant Proteins; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine