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Biotechnol Bioeng. 2004 Apr 20;86(2):188-95.

Enhancing multiple disulfide bonded protein folding in a cell-free system.

Author information

1
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.

Abstract

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.

PMID:
15052638
DOI:
10.1002/bit.10827
[Indexed for MEDLINE]

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