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Applied and Environmental Microbiology, December 2002, p. 5925-5932, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.5925-5932.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

Sriram Srinivasan, Gavin C. Barnard, and Tillman U. Gerngross*

Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755

Received 5 July 2002/ Accepted 10 September 2002

We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.

* Corresponding author. Mailing address: 8000 Cummings Hall, Dartmouth College, Hanover, NH 03766. Phone: (603) 646-3161. Fax: (603) 646-2277. E-mail: tillman.gerngross{at}dartmouth.edu.

Applied and Environmental Microbiology, December 2002, p. 5925-5932, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.5925-5932.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Barnard, G. C., McCool, J. D., Wood, D. W., Gerngross, T. U. (2005). Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha. Appl. Environ. Microbiol. 71: 5735-5742 [Abstract] [Full Text]  
  • Potter, M., Muller, H., Steinbuchel, A. (2005). Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16. Microbiology 151: 825-833 [Abstract] [Full Text]  
  • Potter, M., Muller, H., Reinecke, F., Wieczorek, R., Fricke, F., Bowien, B., Friedrich, B., Steinbuchel, A. (2004). The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. Microbiology 150: 2301-2311 [Abstract] [Full Text]  


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