Folding engineering strategies for efficient membrane production in E. coli

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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.