Polyamine Transport Assay Using Reconstituted Yeast Membranes
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Author:
Date:
2021-01-20
[Abstract] ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.
[摘要] [摘要] ATP13A2 / PARK9是一种晚期内/溶酶体P5B转运ATPase,与多种神经退行性疾病有关。我们最近将ATP13A2表征为溶酶体多胺出口者,这为未知的哺乳动物多胺转运系统的分子身份提供了线索。在这里,我们逐步描述了从过量表达人ATP13A2的酵母细胞中测量重组囊泡中放射性标记的多胺转运的方案。该方案是我们最新出版物的一部分(van Veen等,2020),将有助于表征其他假定的多胺转运蛋白的转运功能,例如P5B转运ATPase的同工型。
[背景] ATP13A2 / PARK9编码一种普遍表达的晚期内-/溶酶体膜蛋白,与一系列神经退行性疾病有关,例如早发性帕金森氏病(Di Fonzo等,2007 ;Lin等,2008)和Kufor -Rakeb综合征(伴痴呆的早期帕金森病)(Ramirez等,2006 ;Park等,2011)。ATP13A2属于P型转运ATPase ,是一类活性转运蛋白,由于ATP水解而暂时形成磷酸中间产物(Kuhlbrandt ,2004年)。ATP13A2是P5亚家族的成员,该家族已在20多年前通过基因组测序鉴定出来(Axelsen和Palmgren ...
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Production, Purification and Characterization of Recombinant Biotinylated Phytochrome B for Extracellular Optogenetics
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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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Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER
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Author:
Date:
2018-01-05
[Abstract] Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and ...
[摘要] 蓝细菌是光合细菌,在不同的生态系统中繁衍,在全球碳循环中发挥重要作用。 蓝藻固定大气CO 2和将固定碳分配到化学品和生物燃料的能力作为可持续的微生物细胞工厂已经引起越来越多的关注。 更好地了解参与中央碳代谢的酶的活性可能导致产物产量增加。 目前,无细胞裂解物被广泛用于细胞内酶活性的测定。 然而,由于蓝细菌细胞壁较厚,蓝细菌细胞的裂解效率低下且费力。 目前的方案描述了一种简单而有效的方法来渗透蓝藻细胞,而不溶解它们,并直接使用透化细胞来测定体内代谢酶活性。
【背景】我们之前已经报道了使用B-PER TM试剂(Thermo Fisher Science)(Rasmussen等人,2016)简单,有效且可扩展的蓝细菌的透化。 B-PER TM TM试剂含有溶解在Tris-HCl缓冲液中的未公开的温和洗涤剂,通常用于裂解细菌细胞如大肠杆菌(Escherichia coli)。偶然地,我们发现B-PER TM TM试剂渗透蓝细菌细胞而不是溶解它们,可能是因为厚的蓝细菌细胞壁(Hoiczyk和Hansel,2000)赋予了试剂中使用的去污剂的抗性。在生物技术感兴趣的蓝细菌中进行通透化。聚球蓝细菌PCC 7002(以下简称“Synechococcus”7002)和“Synechocystis”sp。 PCC ...
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