کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1200009 1493582 2014 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Integrative refolding and purification of histidine-tagged protein by like-charge facilitated refolding and metal-chelate affinity adsorption
ترجمه فارسی عنوان
بازسازی و پاکسازی مجتمع پروتئین برچسب خورده شده با هیستیدین به وسیله شبیه سازی و تسهیل بازسازی و جذب فلزات کلات
کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی


• An integrative method of protein refolding and metal-chelate capture was proposed.
• Iminodiacetic acid modified resins facilitate refolding of like-charged His-tagged protein.
• Addition of nickel ions leads to the affinity capture of the His-tagged protein.
• Facilitated refolding and purification of the His-tagged protein are realized.

This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1 mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1344, 30 May 2014, Pages 59–65
نویسندگان
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