Disulfide Bond Formation in Bacteria-Based Cell-Free Protein Expression
Aaron R. Goerke and James R. Swartz
Cell-free protein synthesis systems have many advantages over conventional in vivo (cellular) expression. For example, they offer the potential for higher productivity, parallel production and simplified purification. Moreover, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make in vitro protein expression systems particularly well suited for the production of patient-specific therapeutic vaccines for diseases such as cancer, for vaccines to protect against threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. Yet the promotion of the cell-free expression system from a useful laboratory tool to a commercially viable technology has been slow. This is at least partially due to the inability to efficiently fold complex proteins, especially those containing disulfide bonds. In this chapter, we describe controlled disulfide/sulfhydryl redox chemistry and improved disulfide isomerization in a cell-free system. We then also describe genetic engineering that in combination results in cell-free systems producing greater complex protein yields than were previously thought possible. To achieve elevated protein yields, cell extracts were generated that maintain amino acid stability and decrease the amount of nonspecific chemical treatment required to eliminate cytoplasmic oxidoreductase activity. In combination, these modifications substantially lower substrate costs since glucose can be used to fuel the cell-free system. We then report the successful scale-up of a B-cell lymphoma fusion vaccine to the 30 mL scale. In considering all of these advances, we believe that our in vitro protein synthesis technology has achieved a significant milestone and is ready for widespread research and commercial implementation.