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Applied and Environmental Microbiology, October 1999, p. 4295-4300, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Stable Escherichia coli-Clostridium acetobutylicum Shuttle Vector for Secretion of Murine Tumor Necrosis Factor Alpha

J. Theys,1 S. Nuyts,2 W. Landuyt,2 L. Van Mellaert,1 C. Dillen,3 M. Böhringer,4 P. Dürre,4 P. Lambin,2 and J. Anné1,*

Laboratories of Bacteriology1 and Immunobiology,3 Rega Institute, Katholieke Universiteit Leuven, and Experimental Radiobiology, University Hospital Gasthuisberg,2 Leuven, Belgium, and Microbiology and Biotechnology, University Ulm, Ulm, Germany4

Received 30 April 1999/Accepted 29 July 1999

Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-alpha ) from Clostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-beta 1,4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-alpha cDNA. The construction was first tested in Escherichia coli and then introduced in C. acetobutylicum DSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-alpha during growth. Significant levels of biologically active mTNF-alpha were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.

* Corresponding author. Mailing address: Laboratory of Bacteriology, Rega Instituut, K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Phone: 3216337371. Fax: 3216337340. E-mail: Jozef.Anne{at}rega.kuleuven.ac.be.

Applied and Environmental Microbiology, October 1999, p. 4295-4300, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.