کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7608500 | 1493382 | 2018 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A two-step purification strategy using calmodulin as an affinity tag
ترجمه فارسی عنوان
یک استراتژی تصفیه دو مرحلهای با استفاده از کالودولین به عنوان یک برچسب تطبیقی
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کلمات کلیدی
تصفیه وابستگی، کالمدولین، دامنه اتصال کامودولین، پروتئین فلورسنت سبز،
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی
Calmodulin (CaM) is a Ca2+-binding protein that plays an important role in cellular Ca2+-signaling. CaM interacts with diverse downstream target proteins and regulates their functions in a Ca2+-dependent manner. CaM changes its conformation and hydrophobicity upon [Ca2+] change and consequently changes its interaction with CaM-binding domains from the targets. Based on these special properties of CaM, it was used as an affinity tag to develop a novel purification strategy by using it for two sequential orthogonal purification steps: 1) an affinity purification step, in which CaM-tag interacts with an immobilized CaM-binding domain; and 2) a hydrophobic interaction chromatography step, during which CaM binds to a phenyl sepharose column. In both steps, the CaM-tagged protein binds in the presence of Ca2+ and unbinds in the presence of ethylenediaminetetraacetic acid (EDTA). An optional third step can be added to remove the CaM-tag if necessary. We used green fluorescent protein (GFP) as a test protein to demonstrate the effectiveness of the method. High yield and high purity of GFP with proper function was obtained using this novel strategy. We believe that this method can be applied to a wide range of protein targets for structural and functional studies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1544, 6 April 2018, Pages 16-22
Journal: Journal of Chromatography A - Volume 1544, 6 April 2018, Pages 16-22
نویسندگان
Lianyun Lin, Chen Liu, Bidhan Chandra Nayak, Weiyi He, Minsheng You, Zhiguang Yuchi,