Folding engineering strategies for efficient membrane production in E. coli

大肠杆菌高效膜生产的折叠工程策略

• 批准号: 0854511
• 负责人: Francois Baneyx
• 金额: $ 40万
• 依托单位: University of Washington
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-15 至 2013-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0854511&HistoricalAwards=false
• 关键词: Folding; engineering; strategies; efficient; membrane

0854511BaneyzIntellectual Merit - E. coli is one of the most popular model organism to study membrane protein biogenesis and one of the most robust and best characterized hosts for heterologous protein production. While membrane proteins can often be expressed as cytoplasmic inclusion bodies in E. coli, their refolding into functional species is challenging and often unsuccessful. Accumulation in membranes circumvents the refolding problem but is usually toxic to the cell, severely reducing yields. Recent genome-wide studies indicating that toxicity effects are associated with translocation machinery saturation, coupled with a growing understanding of the players and processes involved in membrane protein delivery, insertion and folding have opened the door to a new paradigm for membrane protein production that is called folding engineering. Not unlike metabolic engineering, the goal is to optimize the expression host in a holistic fashion to maximize the yields of a desired end product. However, while metabolic engineering addresses the multi-enzyme conversion of a substrate into a high value-added product, folding engineering focuses on the interconnected steps of how a nascent protein is engaged, transferred and folded by molecular chaperones, ushers, insertases, and foldases. Here, the Principal Investigator (PI) proposes to develop and validate transformative folding engineering strategies that will allow efficient membrane protein production in E. coli.Broader Impact - Membrane proteins are found on the surface of every cell and play essential roles in sensing, regulation, cell-to-cell communication and in the binding, import and export of small molecules, peptides and proteins. One subclass of eukaryotic plasma membrane proteins, the G protein-coupled receptors (GPCRs), are the target of over 60% of pharmaceutical drugs currently in use, while bacterial outer membrane proteins are proving increasingly important for the development of much needed antimicrobial agents and vaccines. With applications ranging from disease treatment to photonic devices construction and biofuel production, membrane proteins hold enormous potential in the biotechnology and bionanotechnology sectors. Yet, difficulties associated with their large-scale production have severely hampered fundamental and applied progress. Here, the PI proposes to rely on a growing understanding of the molecular mechanisms of membrane protein trafficking and insertion to develop a holistic folding engineering strategy that will yield enabling tools for the production of both alpha-helical and beta-barrel membrane proteins in E. coli. Graduate students involved in the project will be trained at the interface of molecular biology, biochemistry and engineering and further exposed to the vibrant area of nanobiotechnology. They will gain supervisory experience by overlooking the research of undergraduate and high school students participating in the project and will become involved in the multiple outreach and educational activities conducted by the PI (REU, RET, High School Science for Success program and Nanoethics). Undergraduate students trained in the PI's laboratory (half of which are female) have a track record of joining graduate and medical school programs.

大肠杆菌是研究膜蛋白生物发生的最流行的模式生物之一，也是最健壮和特性最好的异源蛋白生产宿主之一。虽然膜蛋白在大肠杆菌中通常可以以细胞质包涵体的形式表达，但它们重折叠成有功能的物种是具有挑战性的，而且往往不成功。膜上的积累绕过了折叠问题，但通常对细胞有毒性，严重降低产量。最近的全基因组研究表明，毒性效应与转位机制的饱和有关，加上对膜蛋白递送、插入和折叠所涉及的参与者和过程的日益了解，打开了一种新的膜蛋白生产范式的大门，称为折叠工程。与代谢工程不同，目标是以整体的方式优化表达宿主，以最大限度地提高所需最终产品的产量。然而，代谢工程致力于将底物的多酶转化为高附加值的产品，而折叠工程则专注于新生蛋白质如何通过分子伴侣、引座子、插入酶和折叠酶进行参与、转移和折叠的相互关联的步骤。在这里，首席调查员(PI)建议开发和验证变革性折叠工程策略，以便在大肠杆菌中高效地生产膜蛋白。广泛的冲击膜蛋白存在于每个细胞的表面，在传感、调节、细胞间通信以及小分子、多肽和蛋白质的结合、进出口中发挥重要作用。G蛋白偶联受体(GPCRs)是真核质膜蛋白的一个亚类，是目前使用的药物的靶标，而细菌外膜蛋白对开发急需的抗菌剂和疫苗越来越重要。膜蛋白的应用范围广泛，从疾病治疗到光子器件构建和生物燃料生产，在生物技术和生物纳米技术领域具有巨大的潜力。然而，与大规模生产相关的困难严重阻碍了基础性和应用性的进展。在这里，PI建议依靠对膜蛋白运输和插入的分子机制的日益了解来开发一种整体折叠工程策略，该策略将为在大肠杆菌中生产α-螺旋和β-桶状膜蛋白提供可能的工具。参与该项目的研究生将接受分子生物学、生物化学和工程学方面的培训，并进一步接触到充满活力的纳米生物技术领域。他们将通过忽视参与该项目的本科生和高中生的研究来获得指导经验，并将参与PI(REU、RET、高中科学成功计划和纳米伦理)开展的多重外展和教育活动。在PI实验室接受培训的本科生(其中一半是女性)有参加研究生和医学院项目的记录。