کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5914984 1162766 2010 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
NMR characterization of foldedness for the production of E3 RING domains
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شناسی مولکولی
پیش نمایش صفحه اول مقاله
NMR characterization of foldedness for the production of E3 RING domains
چکیده انگلیسی

We summarize the use of NMR spectroscopy in the production and the screening of stability and foldedness of protein domains, and apply it to the RING domains of E3 ubiquitin-ligases. RING domains are involved in specific interactions with E2 ubiquitin-conjugating enzymes and thus play an essential role in the ubiquitination pathway. Protein production of the Zn2+ containing and cysteine rich RING domains for molecular studies frequently turns out to be problematic. We compared the expression and solubility of 14 E3 RING/U-box domains fused to the N-terminal tags of His6, His6-GB1, His6-Trx and His6-GST at small scale and analyzed, by NMR spectroscopy, their correct folding after purification. The addition of GST, Trx or GB1 to the N-terminal His6 tag significantly improved both the expression and solubility of target proteins as compared to His6 tag alone. More importantly most of the immobilized metal affinity chromatography (IMAC) purified proteins were largely unfolded as judged by analysis of the 1H-15N HSQC spectra. We demonstrate that imidazole causes a concentration dependent decrease in stability of RING proteins ascribed to metal depletion and resulting in unfolding or precipitation. In contrast, using glutathione affinity chromatography, the His6-GST fused RING and U-box domains were purified as correctly folded proteins with high yields. Our data clearly demonstrate that IMAC should be avoided and that GST-fusion affinity chromatography is generally applicable for expression and purification of Zn2+ containing proteins.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Structural Biology - Volume 172, Issue 1, October 2010, Pages 120-127
نویسندگان
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