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Applied and Environmental Microbiology, September 2001, p. 3994-4000, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.3994-4000.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Cosecretion of Chaperones and Low-Molecular-Size Medium Additives Increases the Yield of Recombinant Disulfide-Bridged Proteins

Jörg Schäffner, Jeannette Winter, Rainer Rudolph, and Elisabeth Schwarz^*

Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany

Received 22 December 2000/Accepted 3 June 2001

Attempts were made to engineer the periplasm of Escherichia coli to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive L-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 M L-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of E. coli DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm of E. coli.

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^* Corresponding author. Mailing address: Martin-Luther-Universität Halle-Wittenberg, Institut für Biotechnologie, Kurt-Mothes-Str. 3, 06120 Halle, Germany. Phone: 49 345 5524856. Fax: 49 345 5527013. E-mail: elisabeth.schwarz{at}biochemtech.uni-halle.de.

Present address: Scil Proteins GmbH, 06120 Halle, Germany.

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Applied and Environmental Microbiology, September 2001, p. 3994-4000, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.3994-4000.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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