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Applied and Environmental Microbiology, December 1998, p. 4891-4896, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

Ji Qiu,1 James R. Swartz,2 and George Georgiou1,3,*

Molecular Biology Program1 and Department of Chemical Engineering,3 University of Texas, Austin, Texas 78712, and Department of Cell Culture and Fermentation Research and Development, Genentech, Inc., South San Francisco, California 940802

Received 25 June 1998/Accepted 1 September 1998

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 µg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding.

* Corresponding author. Mailing address: Department of Chemical Engineering, University of Texas at Austin, College of Engineering, Austin, TX 78712-1062. Phone: (512) 471-6975. Fax: (512) 471-7963. E-mail: gg{at}che.utexas.edu.

Applied and Environmental Microbiology, December 1998, p. 4891-4896, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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Copyright © 1998 by the American Society for Microbiology. All rights reserved.