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Applied and Environmental Microbiology, October 2005, p. 5735-5742, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5735-5742.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

Gavin C. Barnard,1 Jesse D. McCool,1 David W. Wood,2 and Tillman U. Gerngross1*

Thayer School of Engineering, Department of Biological Sciences, and Department of Chemistry, Dartmouth College, Hanover, New Hampshire, 03755,1 Department of Chemical Engineering, Princeton University, Princeton, New Jersey 085442

Received 24 February 2005/ Accepted 13 May 2005

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active ß-galactosidase was obtained in a single step using the above described method.

* Corresponding author. Mailing address: Thayer School of Engineering, Department of Biological Sciences, and Department of Chemistry, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755. Phone: (603) 646-3161. Fax: (603) 646-2277. E-mail: tillman.gerngross{at}dartmouth.edu.

Applied and Environmental Microbiology, October 2005, p. 5735-5742, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5735-5742.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Neumann, L., Spinozzi, F., Sinibaldi, R., Rustichelli, F., Potter, M., Steinbuchel, A. (2008). Binding of the Major Phasin, PhaP1, from Ralstonia eutropha H16 to Poly(3-Hydroxybutyrate) Granules. J. Bacteriol. 190: 2911-2919 [Abstract] [Full Text]  
  • Brockelbank, J. A., Peters, V., Rehm, B. H. A. (2006). Recombinant Escherichia coli Strain Produces a ZZ Domain Displaying Biopolyester Granules Suitable for Immunoglobulin G Purification. Appl. Environ. Microbiol. 72: 7394-7397 [Abstract] [Full Text]  
  • Peters, V., Rehm, B. H. A. (2006). In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase.. Appl. Environ. Microbiol. 72: 1777-1783 [Abstract] [Full Text]  


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