Detergent-free extraction and purification of membrane proteins to enable structural and functional studies.

膜蛋白的无洗涤剂提取和纯化，以实现结构和功能研究。

• 批准号: BB/J017310/1
• 负责人: Timothy Dafforn
• 金额: $ 54.01万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ017310%2F1
• 关键词: Detergent; free; extraction; purification; membrane

In modern times humanity has benefited from ever improving availability of healthcare and food. These improvements in our quality of life are built upon both scientific and technological innovations, many of which have been made possible by breakthroughs in biochemical sciences. This project aims to capitalise on a recent breakthrough in our laboratory to eventually develop safer and more effective crop protection (CP) compounds and therapeutic drugs. These will in turn increase crop yields, reducing food costs, and improve treatments for diseases, reducing side effects and increasing survival rates.Our ability to develop such chemicals relies on the identification and study or proteins within the cell. More often than not these proteins are found bound to the outer surface of the cell, the membrane, which poses some significant technical difficulties when attempts are made to isolate and study their function. This is in stark contrast to their functional importance in cells, for example, 40% of CP compounds and therapeutic drugs work by interacting with proteins in the membrane. One of the key bottlenecks that limits the study of many cell surface proteins occurs when scientists attempt to release these proteins from the cell surface. This process is essential for investigating the structure of the protein and how it works. The current method for releasing these proteins relies on breaking up the membrane using detergents (not dissimilar to detergents you would find in your kitchen cupboard). The problem with these detergents is that although they are very good at releasing proteins from the cell (we all know how good they are at releasing food, which is high in fat and protein, from a dirty saucepan) they are also very good at scrambling the protein so it no longer functions. This means that the majority of cell surface proteins cannot be released in an active form using these existing methods.In our laboratory we have developed a revolutionary new method that doesn't require detergent to release the protein from the cell surface. Our method is much less harsh than the use of detergent, allowing the production of active proteins. The method is analogous to the way a cookie cutter cuts circular biscuits from a sheet of biscuit dough. We use a simple chemical in place of the biscuit cutter which is able to cut discs (biscuits) from the cell surface (biscuit dough). These discs contain the membrane protein in an active form that can be used in biochemical studies.In this project we aim to make discs that contain proteins that are targets for both crop protection and drug discovery programs. Once we have made these proteins we then aim to develop methods that will allow the structure and function of these proteins to be determined more easily than before. Data from these studies will then be used to improve our ability to develop new drugs and insecticides.

在现代，人类受益于不断改善的医疗保健和食品供应。我们生活质量的这些改善是建立在科学和技术创新的基础上的，其中许多是由于生物化学科学的突破而成为可能的。该项目旨在利用我们实验室最近的突破，最终开发出更安全，更有效的作物保护（CP）化合物和治疗药物。这将反过来增加作物产量，降低食品成本，改善疾病的治疗，减少副作用，提高存活率。我们开发此类化学品的能力依赖于对细胞内蛋白质的识别和研究。这些蛋白质通常被发现结合在细胞的外表面，即细胞膜上，当试图分离和研究它们的功能时，这造成了一些重大的技术困难。这与它们在细胞中的功能重要性形成鲜明对比，例如，40%的CP化合物和治疗药物通过与膜中的蛋白质相互作用而起作用。限制许多细胞表面蛋白质研究的关键瓶颈之一发生在科学家试图从细胞表面释放这些蛋白质时。这个过程对于研究蛋白质的结构及其工作方式至关重要。目前释放这些蛋白质的方法依赖于使用洗涤剂（与您在厨房橱柜中找到的洗涤剂没有什么不同）打破膜。这些清洁剂的问题在于，尽管它们非常擅长从细胞中释放蛋白质（我们都知道它们非常擅长从脏锅中释放富含脂肪和蛋白质的食物），但它们也非常擅长扰乱蛋白质，使其不再起作用。这意味着大多数细胞表面蛋白质不能以活性形式使用这些现有的方法释放。在我们的实验室中，我们开发了一种革命性的新方法，不需要洗涤剂就可以从细胞表面释放蛋白质。我们的方法比使用洗涤剂的苛刻程度要低得多，可以生产活性蛋白质。该方法类似于饼干切割机从饼干面团片上切割圆形饼干的方式。我们使用一种简单的化学品代替饼干切割器，它能够从细胞表面（饼干面团）切割圆盘（饼干）。这些圆盘含有活性形式的膜蛋白，可用于生物化学研究。在本项目中，我们的目标是制造含有蛋白质的圆盘，这些蛋白质是作物保护和药物发现计划的目标。一旦我们制造出这些蛋白质，我们的目标就是开发出能够使这些蛋白质的结构和功能比以前更容易确定的方法。这些研究的数据将用于提高我们开发新药和杀虫剂的能力。

项目成果

Examining the stability of membrane proteins within SMALPs

• DOI: 10.1016/j.eurpolymj.2018.12.008
• 发表时间: 2019-03-01
• 期刊: EUROPEAN POLYMER JOURNAL
• 影响因子: 6
• 作者: Gulamhussein, Aiman A.;Meah, Danyall;Rothnie, Alice J.
• 通讯作者: Rothnie, Alice J.

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.

• DOI: 10.1042/bsr20140171
• 发表时间: 2015-04-16
• 期刊: Bioscience reports
• 影响因子: 4
• 作者: Jamshad M;Charlton J;Lin YP;Routledge SJ;Bawa Z;Knowles TJ;Overduin M;Dekker N;Dafforn TR;Bill RM;Poyner DR;Wheatley M
• 通讯作者: Wheatley M

An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles.

• DOI: 10.1039/c8nr01322e
• 发表时间: 2018-06-07
• 期刊: Nanoscale
• 影响因子: 6.7
• 作者: Hall SCL ;Tognoloni C ;Charlton J ;Bragginton ÉC ;Rothnie AJ ;Sridhar P ;Wheatley M ;Knowles TJ ;Arnold T ;Edler KJ ;Dafforn TR
• 通讯作者: Dafforn TR

Encapsulated membrane proteins: A simplified system for molecular simulation.

• DOI: 10.1016/j.bbamem.2016.02.039
• 发表时间: 2016-03
• 期刊: Biochimica et biophysica acta
• 作者: Sarah C. Lee;S. Khalid;N. Pollock;Timothy J. Knowles;K. Edler;A. Rothnie;Owen R T Thomas;T. Dafforn