Expression And Purification Of Drosophila CRAC Channel Proteins

Ca²⁺ is a major intracellular messenger in eukaryotic cells.Changes in intracellular Ca²⁺ concentration are required for several physiological processes.Store-operated calcium channels?SOCs?constitute the main Ca²⁺ entry pathway into the cells,and the best known and most extensively characterized SOCs are the Ca²⁺-release-activated Ca²⁺?CRAC?channels.CRAC channels control many fundamental cellular functions including gene expressing,cell proliferation and exocytosis.For several decades,the discovery of Orai as a channel protein and STIM as the endoplasmic reticulum?ER?Ca²⁺ sensor is essential for rapid progress in our understanding of the mechanisms and functions of CRAC channels.Upon Ca²⁺ store depletion from ER,STIM with a series of conformational changes and coupled with Orai,resulting in direct protein-protein interaction between the two proteins,and leading to activation of CRAC channels which allow extracellular Ca²⁺ entry into cells.However,little was known of the gating mechanisms of CRAC channels.In order to further reveal the molecular gating mechanism of CRAC channel,we expressed and purified Drosophila STIM protein with different fragments and Orai protein.In the processes,we optimized the separation and purification conditions of STIM protein fragments,detected their functions and ultimately obtained a most stable and functional STIM protein fragments.We also found that the affinity between Orai protein and STIM protein fragment is strong through the determination of the interactions of the two proteins by isothermal titration calorimetry,which provide the basis for further study of the interactions between the two proteins by single-molecule fluorescence resonance energy transfer and co-crystallized protein technology.