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Applied and Environmental Microbiology, October 2002, p. 4979-4985, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4979-4985.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Excretion of Human ß-Endorphin into Culture Medium by Using Outer Membrane Protein F as a Fusion Partner in Recombinant Escherichia coli

Ki Jun Jeong and Sang Yup Lee*

Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon 305-701, Korea

Received 22 April 2002/ Accepted 29 July 2002

Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage {lambda} to construct E. coli MBEL-BL101. For the excretion of human ß-endorphin as a model protein, the ß-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-ß-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-ß-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield ß-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of ß-endorphin was obtained.

* Corresponding author. Mailing address: Department of Chemical and Biomolecular Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea. Phone: 82-42-869-3930. Fax: 82-42-869-8800. E-mail: leesy{at}mail.kaist.ac.kr.

Applied and Environmental Microbiology, October 2002, p. 4979-4985, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4979-4985.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Park, S. J., Lee, S. Y. (2003). Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli. J. Bacteriol. 185: 5391-5397 [Abstract] [Full Text]  
  • Lee, S. Y., Lee, Y. (2003). Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(-)-Hydroxycarboxylic Acids. Appl. Environ. Microbiol. 69: 3421-3426 [Abstract] [Full Text]  


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