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High yield expression and NMR characterization of Arkadia E3 ubiquitin ligase RING-H2 finger domain |
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| Authors: | Nikolaos G Kandias Detlef Bentrop Georgios A Spyroulias |
| Institution: | a Department of Pharmacy, University of Patras, Panepistimioupoli—Rion, GR-26504 Patras, Greece b Institute of Physiology II, University of Freiburg, D-79108 Freiburg, Germany c Mammalian Neurogenesis, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK |
| Abstract: | E3 ubiquitin ligases play a key role in the recognition of target proteins and the degradation by 26S proteasomes. Arkadia is the first example of an E3 ubiquitin ligase that positively regulates TGF-β family signaling. It has been shown to induce ubiquitin-dependent degradation of negative regulators of TGF-β signaling through its C-terminal RING domain. Structural analysis of Arkadia RING domain is needed to elucidate its enzymatic properties. For such studies efficient production of pure and correctly folded Arkadia protein is required. Here we report the recombinant expression in Escherichia coli and purification of the C-terminal RING domain of Arkadia. NMR analysis of the soluble construct reveals a stable folded protein suitable for high resolution structural studies. |
| Keywords: | RING really interesting new gene TGF-β transforming growth factor β Smad mothers against decapentaplegic homolog NMR nuclear magnetic resonance NOESY nuclear overhauser effect spectroscopy HSQC heteronuclear single quantum correlation |
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