| Integral membrane proteins perform many critical functions at the boundaries of cellular compartments, from transporting material across cell membranes to roles in signaling and energy generation. Because of their importance, these proteins are popular targets for research and drug development, making up over 50% of current pharmaceutical targets. However, membrane protein studies have been severely hampered by difficulties in producing sufficient quantities of protein. Conventional in vivo expression strategies generally suffer from cell toxicity, limiting yields to only 0.1-1% of those typically obtained for soluble proteins.; To overcome these obstacles, we have developed an E.coli-based cell-free synthesis system for producing improved yields of integral membrane proteins. Cell-free technology mimics the same transcription and translation reactions that occur in living cells but in an in vitro environment. By supplementing E.coli-derived vesicles, we supply a membrane environment to receive the synthesized protein, and also provide natural translocation machinery to facilitate insertion and folding. Because cell-free systems decouple protein production from cell-growth, they have the potential to accumulate higher yields of toxic proteins compared to in vivo systems. Previously reported in vitro systems have relied on detergent- or lipid-based refolding strategies to generate higher yields. However, our system does not utilize these components, instead activating natural folding pathways to efficiently produce functional membrane proteins.; This thesis describes the development and characterization of a cell-free system supplemented with natural vesicles for membrane protein production. Three bacterial transporters - aquaporin (AqpZ), the tetracycline pump (TetA), and mannitol permease (Mt1A) - were produced in yields of 100-500 mug/mL, up to 400 times typical in vivo yields for these proteins. Proper folding of the cell-free produced protein was verified by protease digestion and activity assays. Taking advantage of the openness of the cell-free environment, we also evaluated factors that limit insertion efficiency in our system, elucidating differences in helper protein dependencies for each model membrane protein. Finally, we describe progress towards adapting this bacterial-based synthesis system for the production of mammalian membrane proteins, in particular strategies for achieving post-translational additions using unnatural amino acids. |
