Author:
Date:
2020-03-05
[Abstract] In the field of extracellular optogenetics, photoreceptors are applied outside of cells to obtain systems with a desired functionality. Among the diverse applied photoreceptors, phytochromes are the only ones that can be actively and reversibly switched between the active and inactive photostate by the illumination with cell-compatible red and far-red light. In this protocol, we describe the production of a biotinylated variant of the photosensory domain of A. thaliana phytochrome B (PhyB-AviTag) in E. coli with a single, optimized expression plasmid. We give detailed instructions for the purification of the protein by immobilized metal affinity chromatography and the characterization of the protein in terms of purity, biotinylation, spectral photoswitching and the ...
[摘要] [摘要 ] 在细胞外光遗传学领域,光感受器被应用于细胞外,以获得具有所需功能的系统。在各种应用的感光体中,植物色素是唯一可以通过与细胞相容的红色和远红外光照射而在活性和非活性光态之间主动和可逆地切换的色素。在此协议中,我们描述了在大肠杆菌中拟南芥光敏色素B(PhyB-AviTag )的光敏域的生物素化变体的生产。 带有一个优化的表达质粒。我们给出了通过固定的金属亲和色谱法纯化蛋白质的详细说明,并根据纯度,生物素化,光谱光开关以及与它的相互作用伴侣PIF6的光依赖性相互作用对蛋白质进行了表征。与以前使用PhyB-AviTag 进行的研究相比,此方案中使用的优化表达质粒简化了生产过程,并显示出更高的产量和纯度。
[背景 ] 在新兴的细胞外光遗传学领域,光感受器被应用于细胞外,例如,以控制生物功能或设计光响应性生物材料(Leung 等,2008;Zhang 等,2015;Chen and Wegner ,2017 ; Lyu 等人,2017 ; Wang 等人,2017 ; Bartelt 等人,2018 ; Beyer 等人,2018a ; Beyer 等人,2018b ; Jia 等人,2018 ; Kolar 等人, 2018 ; Liu 等人,2018 ; Wu 等人,2018 ; Baaske 等人,2019 ; Gil 等人,2019 ; H?rner ...
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