Modern recombinant DNA expression technologies, by definition, challenge cells to engage in unnatural acts; specifically, the production of new types and large quantities of proteins for which the cells have not evolved. Consequently, many natural products are toxic, do not fold properly, or do not fully activate – especially when relatively complex proteins are produced at commercially relevant yields and productivities. The challenge is even more severe when we attempt to produce new proteins or protein assemblies that do not exist in nature. The intracellular environment and catalytic system for protein synthesis and folding are simply not adequate and are difficult to reengineer because of the need to maintain cell viability and because of the barrier imposed by the cell wall.
Cell-free protein synthesis potentially removes these limitations to allow for precise modification and control of the factors influencing protein expression and folding. In spite of its promise, however, cell-free production was slow to develop because early technologies were expensive and difficult to scale to commercial levels. Fortunately, recent advances have now dramatically reduced costs and have enabled convenient scale-up using conventional bioreactors. These advances now place increased importance on exploring and demonstrating the capacity for cell-free methods to produce complex natural proteins that are difficult to produce in vivo. This talk will show how the cell-free system can be manipulated to provide dramatically improved production and folding of several natural proteins that are difficult to produce in vivo because of multiple disulfide bonds, the need to install intrinsic cofactors, or the need to be stabilized by insertion into biological membranes.
New technologies and several examples will also be presented that expand the range of potential products to include a broad range of “supernatural” proteins and protein structures that do not exist in nature but which offer exciting new functionalities. These potential products include complex fusion proteins, proteins containing non-natural amino acids, and large precisely assembled protein complexes to serve as highly effective vaccines, imaging agents, and sophisticated diagnostic reagents.
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