果蝇来源CRAC通道蛋白的表达与纯化

发布时间：2018-05-13 18:25

本文选题：CRAC通道 + Orai　；参考：《四川师范大学》2017年硕士论文

【摘要】：Ca~(2+)是真核细胞重要的细胞内信使。细胞内Ca~(2+)浓度的变化是许多生理进程所必需的。钙库操纵的钙通道(SOCs)是钙离子流入细胞内的主要的方式,其中最著名最具特色的是钙离子释放激活钙离子(CRAC)通道。CRAC通道调节许多基本的细胞功能,包括基因表达、细胞增殖、细胞分泌等。在近十几年里,作为CRAC通道的Orai蛋白和作为内质网钙离子感受器的STIM蛋白的发现是理解CRAC通道的机制和功能取得极大进步的基础。当内质网钙库释放后,STIM蛋白发生一系列的构象变化与Orai蛋白相互作用,从而开放CRAC通道,允许细胞外的钙离子流入细胞内。但CRAC通道门控的分子机制仍然知之甚少。为了进一步揭示CRAC通道门控的分子机制,我们表达并分离纯化了果蝇源的Orai蛋白和STIM蛋白不同片段。在过程中对STIM蛋白不同片段的分离纯化条件进行了优化,对STIM蛋白不同片段进行了功能测试,并最终获得了较稳定并具有功能的STIM蛋白片段,同时对纯化的Orai蛋白和STIM蛋白片段进行了等温滴定测定其相互作用。我们发现Orai蛋白与STIM蛋白片段亲和力较强,这为后续通过单分子荧光能量转移技术和蛋白技术共结晶进一步的研究两蛋白的相互作用的构象变化奠定了基础。
[Abstract]:Ca~(2) is an important intracellular messenger in eukaryotic cells. Changes in intracellular Ca~(2 concentration are necessary for many physiological processes. Calcium channel SOCsis the main way of calcium ions flowing into cells. The most famous and characteristic is calcium ion release activated calcium ion CRAC.CRAC channel regulates many basic cellular functions, including gene expression and cell proliferation. Cell secretion, etc. In recent years, the discovery of Orai protein as CRAC channel and STIM protein as endoplasmic reticulum calcium receptor has been the basis of great progress in understanding the mechanism and function of CRAC channel. When the endoplasmic reticulum calcium reservoir was released, a series of conformational changes occurred between STIM protein and Orai protein, thus opening up the CRAC channel and allowing extracellular calcium ions to flow into the cell. However, little is known about the molecular mechanism of CRAC channel gating. In order to further reveal the molecular mechanism of CRAC channel gating, we expressed and purified different fragments of Orai and STIM proteins from Drosophila melanogaster. In the process, the conditions of isolation and purification of different STIM protein fragments were optimized, and the functional tests of different STIM protein fragments were carried out. Finally, stable and functional STIM protein fragments were obtained. At the same time, the interaction between purified Orai protein and STIM protein was determined by isothermal titration. We found that Orai protein has strong affinity with STIM protein fragment, which lays a foundation for further study on conformation change of interaction between two proteins by single molecule fluorescence energy transfer technique and protein technology.
【学位授予单位】：四川师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51

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