We describe the genetically directed incorporation of aminoxy functionality into recombinant proteins using a mutant Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNACUA pair. This allows the general production of non-hydrolyzable ubiquitin conjugates of recombinant origin via bioorthogonal oxime ligation. This is exemplified by the preparation of non-hydrolyzable versions of diubiquitin, polymeric ubiquitin chains and ubiquitinated SUMO. We demonstrate that the conjugates exhibit unrivalled isostery with the native isopeptide bond through both structural and biophysical characterization. Furthermore, the conjugates function as nanomolar inhibitors of deubiquitinating enzymes and are recognized by linkage-specific antibodies. This technology should provide a versatile platform for the development of powerful research tools for studying deubiquitinating enzymes and for defining the cellular roles of diverse polyubiquitin linkages.