ABSTRACT
C ell-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 o f the cell-free system allows control o f the reaction environment to promote folding o f disulfide bonded proteins in a rapid and economically feasible format. These advantages make in vitro protein expression systems particularly well suited for the production o f 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 o f 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 efficiendy 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 gener ated that maintain amino acid stability and decrease the amount o f 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 successfid scale-up o f a B-cell lymphoma fusion vaccine to the 30 mL scale. In consider ing all o f 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.
