期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1544, 期 -, 页码 16-22出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2018.02.045
关键词
Affinity purification; Calmodulin; Camodulin-binding domain; Green fluorescent protein
资金
- National Key R&D Program of China [2017YFD0201400, 2017YFD0201403]
- National Nature Science Foundation of China [31320103922, 31230061]
- Project of National Basic Research (973) Program of China [2015CB856500, 2015CB856504]
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. (C) 2018 Elsevier B.V. All rights reserved.
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