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Genetically Directed Production of Recombinant, Isosteric and Nonhydrolysable Ubiquitin Conjugates
Author(s) -
Stanley Mathew,
Virdee Satpal
Publication year - 2016
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201600138
Subject(s) - recombinant dna , ubiquitin , biochemistry , bioorthogonal chemistry , protein engineering , chemistry , conjugate , enzyme , biology , click chemistry , combinatorial chemistry , gene , mathematical analysis , mathematics
We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl‐tRNA synthetase/tRNA CUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage‐specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages.