A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30degreesC increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 mug/mL of bioactive PA in a simple 3-h batch reaction. (C) 2004 Wiley Periodicals Inc.