| 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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| Lectin Binding Analysis of Streptococcus mutans Glycoproteins
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Author:
Date:
2015-04-05
[Abstract] Bacterial glycoproteins are of increasing interest due to their abundance in nature and importance in health and infectious diseases. However, only a very small fraction of bacterial glycoproteins have been characterized and its post-translational modification machinery identified. While analysis of glycoproteins can be achieved through various techniques, this is often limited by the specific characteristics of individual proteins such as type and level of glycosylation. Lectins are sugar-binding proteins that recognize specific glycoconjugates in a manner similar to antigen-antibody interactions. Here, we describe a simple method for the detection of glycoproteins using lectin-based Western blot analysis, which can be applied to different organisms and coupled with various other ...
[摘要] 细菌糖蛋白由于其在自然界中的丰富性和在健康和传染病中的重要性而日益受到关注。 然而，只有非常小部分的细菌糖蛋白已被表征和其翻译后修饰机制鉴定。 尽管通过各种技术可以实现糖蛋白的分析，但是这常常受到单个蛋白质的特定特征的限制，例如糖基化的类型和水平。 凝集素是以类似于抗原 - 抗体相互作用的方式识别特异性糖缀合物的糖结合蛋白。 在这里，我们描述了一种简单的方法使用基于凝集素的蛋白质印迹分析检测糖蛋白，这可以应用于不同的生物体，并与各种其他策略的补充分析。
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