Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli
Article first published online: 25 NOV 2003
Copyright © 2003 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 85, Issue 2, pages 122–129, 20 January 2004
How to Cite
Kim, D.-M. and Swartz, J. R. (2004), Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol. Bioeng., 85: 122–129. doi: 10.1002/bit.10865
- Issue published online: 29 DEC 2003
- Article first published online: 25 NOV 2003
- Manuscript Accepted: 25 AUG 2003
- Manuscript Received: 24 SEP 2002
- cell-free protein synthesis;
- disulfide bond formation;
- protein folding;
- sulfhydrylredox potential
In this report, we demonstrate that a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system. Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions. The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 μg/mL of active urokinase protease in a simple batch reaction. This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach. © 2003 Wiley Periodicals, Inc.