Enhancing multiple disulfide bonded protein folding in a cell-free system.
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 30 degrees C 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 microg/mL of bioactive PA in a simple 3-h batch reaction.
Yin
Gang
G
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Swartz
James R
JR
D002474:Cell-Free System / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000737:chemistry*; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D017510:Protein Folding
D004220:Disulfides; D010960:Plasminogen Activators; D019704:Protein Disulfide-Isomerases; D007460:Iodoacetamide