crystal-structure-n-terminal-domain-ryanodine-receptor-from-plutella

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from <em>Plutella xylostella</em>

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Tianjin University

Calmodulin (CaM) is a Ca-binding protein that plays an important role in cellular Ca-signaling. CaM interacts with diverse downstream target proteins and regulates their functions in a Ca-dependent manner. CaM changes its conformation and hydrophobicity upon [Ca] 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 Ca 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.

Bidhan Chandra Nayak

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