Folding engineering strategies for efficient membrane production in E. coli

0854511 Baneyz智力成绩- E.大肠杆菌是研究膜蛋白生物合成的最流行的模式生物之一，也是生产异源蛋白的最稳定和最具特征的宿主之一。 而膜蛋白在大肠杆菌中常以胞质包涵体形式表达。在大肠杆菌中，它们重折叠成功能性物种是具有挑战性的，并且通常不成功。在细胞膜中的积累可以避免重折叠问题，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，毒性效应与易位机制饱和有关，再加上对膜蛋白递送、插入和折叠中所涉及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，这种新范式被称为折叠工程。与代谢工程不同，其目标是以整体方式优化表达宿主，以最大限度地提高所需终产物的产量。然而，虽然代谢工程解决了将底物转化为高附加值产品的多酶转化，但折叠工程侧重于新生蛋白质如何通过分子伴侣、引导子、插入酶和折叠酶接合、转移和折叠的相互关联的步骤。在这里，主要研究者（PI）建议开发和验证变革性折叠工程策略，这将允许在大肠杆菌中高效生产膜蛋白。广泛的影响-膜蛋白存在于每个细胞的表面，在传感、调节、细胞间通讯以及小分子、肽和蛋白质的结合、输入和输出中发挥重要作用。真核细胞质膜蛋白的一个亚类，G蛋白偶联受体（GPCR），是目前使用的超过60%的药物的靶标，而细菌外膜蛋白被证明对于开发急需的抗微生物剂和疫苗越来越重要。随着从疾病治疗到光子器件构建和生物燃料生产的应用，膜蛋白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本和实用的进展。在这里，PI建议依赖于对膜蛋白运输和插入的分子机制的不断了解，以开发一种整体折叠工程策略，该策略将产生用于在大肠杆菌中生产α螺旋和β桶膜蛋白的工具。杆菌参与该项目的研究生将在分子生物学，生物化学和工程的接口进行培训，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监督经验，并将参与PI（REU，RET，高中科学成功计划和纳米伦理学）进行的多种推广和教育活动。在PI实验室培训的本科生（其中一半是女性）有参加研究生和医学院课程的记录。

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.