High yield expression and NMR characterization of Arkadia E3 ubiquitin ligase RING-H2 finger domain.
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-beta family signaling. It has been shown to induce ubiquitin-dependent degradation of negative regulators of TGF-beta 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.
Kandias
Nikolaos G
NG
Department of Pharmacy, University of Patras, Panepistimioupoli-Rion, GR-26504 Patras, Greece.
Chasapis
Christos T
CT
Bentrop
Detlef
D
Episkopou
Vasso
V
Spyroulias
Georgios A
GA
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D054829:RING Finger Domains; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D025801:Ubiquitin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015032:Zinc / Q000737:chemistry
D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1; D025801:Ubiquitin; C427283:Rnf111 protein, mouse; D044767:Ubiquitin-Protein Ligases; D015032:Zinc