Intellectual 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.
Background: Membrane proteins play essential role in cell physiology and survival, have been implicated in many human diseases, and are the target of 60% of all pharmaceutical drugs (a $50B market). They also hold enormous promise in the burgeoning bionanotechnology area where they can be used as sensors, transporters, transducers, motors, and for the construction of novel nano-electronic and optical devices. Unfortunately, little is known about the structure and function of membrane proteins and their technological use has been limited because they often misfold and kill cells when one attempts to produce them in any significant amount. Intellectual Merit: The goal of this project was to improve the current understanding of the roles of folding modulators in the biogenesis of membrane proteins in E. coli, and to use this knowledge to construct bacterial strains and plasmids allowing for efficient production of membrane and secretory proteins in a biologically active form. We were able to show that: Strains carrying a deletion in the gene encoding chaperone Trigger Factor can improve the production of functional α-helical membrane proteins by 3 to 7-fold, leading to yields in the tens of mg/L. The same strains are suitable for doubling the yields of leech carboxypeptidase inhibitor, a small disulfide-bonded protein that holds promise for the treatment of cardiovascular diseases. A spontaneous mutant in a genetic switch used to control recombinant membrane protein production (the PBAD promoter) can significantly enhances α-helical membrane protein yields, particularly when used in combination with trigger factor deficient cells. Co-expression of the chaperone YidC improves the yields of α-helical membrane proteins containing multiple membrane-spanning segments and that a truncated gain of function YidC mutant is particularly effective at the task Tandem repeat of standard signal sequences are functional for the export of β-barrel outer membrane proteins and reduce the accumulation of misfolded species. Broader Impact. The strains and plasmids generated in this work have so far been shared with 11 US and international laboratories working in membrane protein expression. The award supported the publication of 5 journal articles and 2 additional papers are in the works. Graduate (3) and undergraduate (1) students involved in this project have been trained at the interface of molecular biology, biochemistry and molecular biology. Students and PI conducted K-12 and public outreach activities to introduce learners of all ages to the field of biotechnology. Research results have been incorporated in the Biochemical Engineering course and educational modules.taught by the PI.
