Expression and Purification of Arabidopsis Transmembrane Protein BCM1 in Saccharomyces cerevisiae
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Author:
Date:
2020-09-20
[Abstract] Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1) showed a stimulatory effect of the BCM1 on magnesium chelatase, the first enzyme of chlorophyll biosynthesis, through interaction with the GENOMES UNCOUPLED 4 (Wang et al., 2020). Here, we report a detailed and optimized method for heterologous expression and purification of His-tagged BCM1 in Saccharomyces cerevisiae. Following this method, we obtained native BCM1 used for in vitro ...
[摘要] [摘要 ] 异源表达和公顷跨膜蛋白的纯化VE 仍然几十年来阻碍了关键酶详述生物化学和结构表征一个挑战小号和它们的相互作用调节在多个代谢途径。上新鉴定进行了深入的研究拟南芥拟南芥叶绿素代谢1(BCM1)的整合膜蛋白BALANCE显示一个通过与相互作用对镁螯合,叶绿素生物合成的第一个酶,所述BCM1的刺激效应基因组中脱开4 (王等等人,2020)。这里 ,我们报告了酿酒酵母中His-tagged BCM1异源表达和纯化的详细和优化方法。˚F ollowing这种方法,我们获得用于本机BCM1 体外酶测定的镁螯合(王等人,2020) 。目前,BCM1的结晶研究正在进行中。这个协议可以适于纯化BCM 1一样从用于酶和结构研究真核生物的跨膜蛋白。
[背景 ] 鉴定翻译后单组的lators其指导LY 调制enzym 一个叶绿素合成的酶的抽动活动可以大大提高我们理解的分子机制,通过该植物保持高效叶绿素叶期间LL合成绿化(Brzezowski 等人,2015年)。然而,叶绿素合成酶及其相互作用蛋白的详细生化分析受到体外重组蛋白可用性的限制。我们最近发现一个叶绿素代谢1(BCM1)的翻译后调节平衡,同时刺激小号叶绿素合成和延迟叶绿素分解,日ERE 被授予叶发育过程中的叶绿素稳态(王等人,2020年)。为了检查BCM1对镁螯合酶(MgCh ...
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Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation
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Author:
Date:
2020-05-20
[Abstract] Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the mechanism of histone transport and also the potential roles of importins as histone chaperones and in the assembly of nucleosomes. Here we report a simple and reproducible sedimentation-velocity based method to determine the molecular sizes of importin-histone complexes using analytical ultracentrifugation. The method does not use any reporter tags or interaction with column resin thereby analyzing the interactions of the native proteins.
[摘要] [摘要] 组蛋白构成核小体的蛋白质成分。尽管其尺寸很小,但组蛋白不会通过核孔复合物扩散。取而代之的是,它们单独或与组蛋白分子伴侣复合地被重要蛋白转运至细胞核。确定importin-histone复合物的分子大小是理解组蛋白转运机制的关键,也是importins作为组蛋白伴侣和在核小体组装中的潜在作用的关键。在这里,我们报告了一种简单且可重现的沉降速度为基础的方法,该方法使用分析超速离心法来确定importin-histone配合物的分子大小。该方法不使用任何报告子标签或与色谱柱树脂的相互作用,从而分析了天然蛋白质的相互作用。
[背景] 核小体是真核染色质的最基本的结构和功能单元。组蛋白H2A,H2B,H3和H 4是核小体的蛋白质成分。每个核包括147个碱基的DNA wrapp的对编绕Ñ H3-H4四聚体和H2A-H2B二聚体的两个拷贝(Luger的等人,1997年一)。像细胞中的其他蛋白质一样,组蛋白在细胞质中合成。然而,核小体组装在核中。尽管它们的小尺寸(单体是10-15 kDa)的,组蛋白不通过核孔复合物扩散,而是可以单独使用或在复合物与由组蛋白importins伴侣输送要么(约翰逊-SAL IBA 等人,2000 ; Baake 等等人,2001;Mosammaparast 等人,2001,2002a和2002b;Muhlhausser ...
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Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification
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Author:
Date:
2018-11-20
[Abstract] Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer’s. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the functions of Tau and the generation of pharmacological agents targeting Tau has been the difficulty of obtaining soluble Tau protein in purified form. Here, we describe a protocol that uses dual affinity tag purification to selectively purify soluble recombinant Tau protein from bacteria that is functionally active for downstream applications including immunization, microtubule binding assays, and protein-protein interaction studies.
[摘要] 微管相关蛋白Tau(由 MAPT 基因编码)的功能障碍已经涉及20多种神经退行性疾病,包括阿尔茨海默病。 因此,Tau的生理和疾病相关功能引起了研究界的极大兴趣。 妨碍对Tau功能的研究和产生靶向Tau的药理学试剂的一个障碍是难以获得纯化形式的可溶性Tau蛋白。 在这里,我们描述了一种方案,该方案使用双亲和标签纯化从细菌中选择性纯化可溶性重组Tau蛋白,所述细菌对于下游应用具有功能活性,包括免疫,微管结合测定和蛋白质 - 蛋白质相互作用研究。 【背景】Tau传统上被定义为微管结合蛋白;然而,在人类疾病中,Tau可以与轴突微管分离并错误定位到其他神经元区室,包括体细胞,树突和突触,其中与非微管蛋白和结构的相互作用驱动神经元功能障碍(Iqbal et al。 ,2016; Wang和Mandelkow,2016; Zhou et al。,2017; McInnes et al。,2018)。尽管神经原纤维缠结形式的Tau聚集体通常存在于死后患病的脑组织中,但研究表明,可溶性Tau,而不是聚集的Tau,是神经元功能障碍的主要原因(Crimins et al。,2012 ; Polydoro et al。,2014; Koss et al。,2016)。因此,研究Tau在疾病中的可溶性功能,例如鉴定蛋白质 - ...
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