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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High-throughput YO-PRO-1 Uptake Assay for P2X7 Receptors Expressed in HEK Cells
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Author:
Date:
2018-07-20
[Abstract] P2X7 receptors are extracellular ATP-gated ion channels that play broad physiological and pathological roles in animals (Sluyter, 2017). Activation of P2X7 receptors lead to the opening of membrane channels permeable for small cations like Na+ and Ca2+ as well as fluorescent dyes such as YO-PRO-1 (Alves et al., 2014). Taking advantage of this dye-permeability, YO-PRO-1 uptake assays have been widely used to probe P2X7 receptor activity (Surprenant et al., 1996; Rassendren et al., 1997; Karasawa et al., 2017). Here we describe a step by step protocol for a high-throughput YO-PRO-1 uptake assay using HEK293 cells expressing P2X7 receptors. This 3-day protocol is particularly suited for examining effects of small molecules and ...
[摘要] P2X7受体是细胞外ATP门控离子通道,在动物中发挥广泛的生理和病理作用(Sluyter,2017)。 P2X7受体的激活导致膜通道的开放可渗透小阳离子如Na + 和Ca 2 + 以及荧光染料如YO-PRO-1(Alves) et al。,2014)。 利用这种染料渗透性,YO-PRO-1摄取试验已被广泛用于探测P2X7受体活性(Surprenant et al。,1996; Rassendren et al。 ,1997; Karasawa et al。,2017)。 在这里,我们描述了使用表达P2X7受体的HEK293细胞进行高通量YO-PRO-1摄取测定的逐步方案。 这个为期3天的方案特别适用于检查小分子和突变对P2X7受体功能的影响。 该协议改编自我们之前发表的论文(Karasawa和Kawate,2016)。
【背景】P2X7受体打开可渗透阳离子的膜通道(Surprenant et al。,1996; Sluyter,2017)。虽然电生理学仍然是量化P2X7受体活性的金标准,但这种专门技术可能并不容易获得。此外,电生理学不适合高通量筛选,因为每次记录需要大量的时间和操作。因此,荧光分子的摄取是一种广泛使用的替代方法,特别是用于筛选多种条件/突变体(Cankurtaran-Sayar et al。,2009; Qu et al。 ...
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