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Enhancing multiple disulfide bonded protein folding in a cell‐free system
Author(s) -
Yin Gang,
Swartz James R.
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10827
Subject(s) - cell free protein synthesis , chemistry , periplasmic space , protein disulfide isomerase , protein folding , biochemistry , polyethylene glycol , spermidine , glutathione , bioprocess , chaperone (clinical) , iodoacetamide , putrescine , disulfide bond , combinatorial chemistry , cysteine , escherichia coli , enzyme , protein biosynthesis , chemical engineering , medicine , pathology , engineering , gene
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°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 μg/mL of bioactive PA in a simple 3‐h batch reaction. © 2004 Wiley Periodicals, Inc.