| Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
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Author:
Date:
2017-09-05
[Abstract] The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is ...
[摘要] P2X7受体是仅在真核生物中发现的胞外ATP门控离子通道（Bartlett等，2014）。由于其P2X受体之间的独特性质，例如大电导孔的形成，P2X7受体已经涉及破坏性疾病如慢性疼痛（North和Jarvis，2013）。然而，P2X7特异性属性的机制仍然知之甚少，部分原因是纯化这种真核膜蛋白是一个挑战。在这里，我们描述了使用昆虫细胞 - 杆状病毒系统表达和纯化哺乳动物P2X7受体的详细方案。 P2X7受体在作为GFP融合蛋白的Sf9昆虫细胞中表达，并用含有Triton X-100洗涤剂的缓冲液溶解。然后使用Strep-Tactin亲和层析在含有十二烷基麦芽糖苷的缓冲液中纯化P2X7-GFP融合蛋白。在通过凝血酶酶切割连接的GFP和Strep-标签后，使用大小排阻色谱分离P2X7受体。该方法通常从6L的Sf9培养物产生约2mg的纯化蛋白质。纯化的蛋白质可以用含有15％甘油的缓冲液在4℃下储存至少2个月，并用于各种功能和结构研究（Karasawa和Kawate，2016）。
【背景】P2X7受体是嘌呤能P2X受体家族的七种亚型之一，并且是广泛疾病如神经退行性疾病，癫痫和神经性疼痛的有希望的新型药物靶点（North和Jarvis，2013; Bhattacharya和Biber， ...
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| Axenic Culture of Sclerotinia sclerotiorum and Preparation of Sclerotinia Culture Filtrate Elicitor 1 (SCFE1)-containing Fractions, Triggering Immune Responses in Arabidopsis thaliana
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Author:
Date:
2014-09-05
[Abstract] The necrotrophic white mold fungus Sclerotinia sclerotiorum (S. sclerotiorum) is pathogenic to a broad range of plant species, including the Brassicaceae model plant Arabidopsis thaliana (Boland and Hall, 1994; Bolton et al., 2006). In Arabidopsis thaliana (A. thaliana), the semi-purified proteinaceous S. sclerotinorum elicitor SCFE1 (Sclerotinia culture filtrate elicitor 1) is sensed at the plasma membrane by the receptor-like protein RLP30 and triggers strong immune responses (Zhang et al., 2013), similar to the bacterial elicitor flagellin (Felix et al., 1999). Elicitation of plant defenses with SCFE1 is a tool to dissect the signaling pathway involving RLP30 and to study immunity to necrotrophic ...
[摘要] 坏死性白霉菌真菌（Sclerotinia sclerotiorum）（核盘菌）对广泛的植物物种（包括十字花科模式植物拟南芥（Arabidopsis thaliana））具有致病性， （Boland和Hall，1994; Bolton等人，2006）。在拟南芥中（thaliana），是半纯化的蛋白质。通过受体样蛋白RLP30在质膜感测核盘菌诱导物SCFE1（核盘菌属培养物滤液诱发物1），并且引发强烈的免疫应答（Zhang等人。，2013），类似于细菌激发剂鞭毛蛋白（Felix等人，1999）。植物防御与SCFE1的诱导是一个工具，以解剖涉及RLP30的信号通路和研究对坏死性真菌的免疫。在这里，我们描述一个简单的协议来生长。菌核。此外，我们提出了一种两步液相色谱法从培养滤液中部分纯化SCFE1的方法（图1A-B）。通过气相色谱法测定植物应激激素乙烯的发射作为生物测定，以在整个纯化程序中监测级分中的激发剂活性（图1C）。
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