Abstract
The use of cell‐free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, 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 cell‐free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man‐made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell‐free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli‐based CFPS system. Two unique CFPS platforms were established by developing standardized extract preparation protocols and generic cell‐free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 µg/mL, an antibody fragment at 230 µg/mL, and a vaccine fusion protein at 300 µg/mL; with the majority correctly folded. Better yields were obtained when cell‐free reaction conditions were optimized for each protein. Establishing general CFPS platforms enhances the potential for cell‐free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines. Biotechnol. Bioeng. 2008;99: 351–367. © 2007 Wiley Periodicals, Inc.