Developing plants as an expression system for mammalian ion channels

膜蛋白是目前市场上大约三分之二的药物的靶点，它们在动物和植物的感染和免疫中起着关键作用。如果可以确定更多的人膜蛋白结构，药物开发的进展将大大加快，但这是一个重大的挑战;当与真核细胞膜蛋白一起工作时遇到的许多问题之一是低表达产率，特别是正确折叠和稳定的蛋白质的低产率。目前最好的真核细胞膜蛋白表达系统（哺乳动物和昆虫细胞培养）使用昂贵，易于感染，因此开发一种广泛可用且廉价的系统将具有显著的益处。植物代表了这一挑战的潜在解决方案;它们种植成本低，产量高，并且通过使用拟南芥或本塞姆氏烟草等模型实验系统，已经有大量的分子工具来简化蛋白质的克隆和表达。本项目的主要目的是开发真核细胞膜蛋白的植物表达系统，重点是ATP门控离子通道的P2 X受体家族。P2 X受体存在于脊椎动物中，它们感知细胞外ATP并发挥各种生理作用，包括疼痛和味觉，控制血管张力和先天免疫。人体内有7种P2 X受体亚型，它们是潜在镇痛和抗炎药物的重要靶点，但迄今为止，仅确定了一种人P2 X受体晶体结构（P2 X3）。了解P2 X受体3D结构将使我们能够设计亚型选择性小分子调节剂，这可能具有显着的治疗益处，特别是在与年龄相关的炎症性疾病，如关节炎，动脉粥样硬化和黄斑变性。该项目是一个令人兴奋的机会，培训膜蛋白表达和植物遗传学专家之间的新合作，学习和应用各种技术，包括分子克隆，转基因植物产生，植物培养，细胞培养，蛋白质表达，蛋白质纯化和使用荧光成像的功能测定。成功将导致新的工具的开发，使生产大量的纯，折叠膜蛋白的直接结构和功能的研究，并显着有利于膜蛋白结构生物学领域。

Membrane proteins are the target for approximately two thirds of currently marketed pharmaceuticals, and they play critical roles in infection and immunity in both animals and plants. Progress in drug development would be substantially accelerated if more human membrane protein structures could be determined, but this is a significant challenge; one of the many problems encountered when working with eukaryotic membrane proteins is low expression yield, in particular a low yield of correctly folded and stable protein. The best currently available expression systems for eukaryotic membrane proteins (mammalian and insect cell culture) are expensive to use and prone to infection, and so the development of a widely available and cheap system would be of significant benefit. Plants represent a potential solution to this challenge; they are cheap to grow, they can produce massive yields, and, by using a model experimental system such as Arabidopsis thaliana or Nicotiana benthamiana, there are a vast array of molecular tools already in place to simplify the cloning and expression of proteins. The main aim of this project is to develop plant expression systems for eukaryotic membrane proteins, focusing on the P2X receptor family of ATP-gated ion channels. P2X receptors are found in vertebrates, where they sense extracellular ATP and play a wide variety of physiological roles including pain and taste sensation, control of blood vessel tone and innate immunity. There are seven P2X receptor subtypes in humans, and they are important targets for potential analgesic and anti-inflammatory drugs, but to date, only one human P2X receptor crystal structure has been determined (P2X3). Understanding P2X receptor 3D-structures would enable us to design subtype-selective small-molecule modulators, which may have significant therapeutic benefit, particularly in age-related inflammatory diseases such as arthritis, atherosclerosis and macular degeneration. This project is an exciting opportunity to train in a new collaboration between experts in membrane protein expression and plant genetics, learning and applying a variety of techniques including molecular cloning, transgenic plant generation, plant culture, cell culture, protein expression, protein purification and functional assays using fluorescence imaging. Success would lead to the development of new tools to enable the production of large quantities of pure, folded membrane proteins for direct structural and functional studies, and be of significant benefit to the field of membrane protein structural biology.