Folding engineering strategies for efficient membrane production in E. coli

大肠杆菌是研究膜蛋白生物发生最流行的模式生物之一，也是最强大和最具特征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表达为细胞质包涵体，但它们的再折叠是具有挑战性的，而且往往是不成功的。在膜上的积累绕过了再折叠问题，但通常对细胞有毒，严重降低产量。最近的全基因组研究表明，毒性效应与转运机制饱和有关，再加上对膜蛋白传递、插入和折叠的参与者和过程的日益了解，为膜蛋白生产的新范式打开了大门，这一范式被称为折叠工程。与代谢工程不同，目标是以整体方式优化表达宿主，以最大限度地提高所需最终产品的产量。然而，代谢工程解决了多酶将底物转化为高附加值产品的问题，而折叠工程关注的是新生蛋白质如何被分子伴侣、引子、插入酶和折叠酶参与、转移和折叠的相互联系的步骤。在这里，首席研究员（PI）建议开发和验证转化折叠工程策略，这将允许在大肠杆菌中高效地生产膜蛋白。更广泛的影响-膜蛋白存在于每个细胞表面，在传感、调节、细胞间通讯以及小分子、肽和蛋白质的结合、输入和输出中起着至关重要的作用。真核质膜蛋白的一个亚类，G蛋白偶联受体（gpcr），是目前使用的60%以上药物的靶标，而细菌外膜蛋白对于开发急需的抗菌剂和疫苗越来越重要。膜蛋白的应用范围从疾病治疗到光子器件的构建和生物燃料的生产，在生物技术和生物纳米技术领域具有巨大的潜力。然而，与大规模生产有关的困难严重阻碍了基础和应用方面的进展。在这里，PI建议依靠对膜蛋白运输和插入的分子机制的不断增长的理解来开发一个整体的折叠工程策略，这将为在大肠杆菌中生产α -螺旋和β -桶膜蛋白提供有利的工具。参与该项目的研究生将在分子生物学，生物化学和工程学的界面上接受培训，并进一步接触充满活力的纳米生物技术领域。他们将通过监督参与项目的本科生和高中生的研究获得管理经验，并将参与PI （REU， RET，高中科学成功计划和纳米伦理）开展的多种推广和教育活动。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。

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.