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标，它们在动植物的感染和免疫中发挥着关键作用。如果能够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；在处理真核膜蛋白时遇到的许多问题之一是低表达产量，特别是正确折叠和稳定的蛋白质产量低。目前最好的真核生物膜蛋白表达系统(哺乳动物和昆虫细胞培养)都是昂贵的，而且容易感染，因此开发一种广泛可用的廉价系统将是非常有益的。植物代表着这一挑战的潜在解决方案；它们种植成本低，可以产生大量产量，并且通过使用拟南芥或本特米纳烟草等模型实验系统，已经有大量分子工具到位，以简化蛋白质的克隆和表达。该项目的主要目的是开发真核膜蛋白的植物表达系统，重点是ATP门控离子通道的P2X受体家族。在脊椎动物中发现了P2X受体，它们感知细胞外的ATP，发挥着广泛的生理作用，包括痛觉和味觉、血管张力的控制和先天免疫。人类有7种P2X受体亚型，它们是潜在的镇痛和抗炎药物的重要靶点，但到目前为止，只确定了一种人类P2X受体的晶体结构(P2X3)。了解P2X受体的三维结构将使我们能够设计具有亚型选择性的小分子调节剂，这些调节剂可能具有显著的治疗益处，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等方面。这个项目是一个令人兴奋的机会，可以在膜蛋白表达和植物遗传学专家之间的新合作中进行培训，学习和应用各种技术，包括分子克隆、转基因植物生成、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进行功能分析。该技术的成功将导致新的工具的开发，使之能够大量生产用于直接结构和功能研究的纯折叠膜蛋白，并将对膜蛋白结构生物学领域产生重大的益处。

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.