Applied Microbiology and Biotechnology

, Volume 46, Issue 5, pp 524–532

High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions

Authors

  • U. Horn
    • Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstraße 11, D-07745 Jena, Germany Fax: + 0049 3641 65 6800 E-mail: diersenb@leutra.imb-jena.de
  • W. Strittmatter
    • Merck KGaA, Med Chem Bioorganica, Frankfurter Straße 250, D-64271 Darmstadt, Germany
  • A. Krebber
    • Biochemisches Institut der Universität Zürich, Winterthurer Str. 190, CH-8057 Zürich, Switzerland
  • U. Knüpfer
    • Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstraße 11, D-07745 Jena, Germany Fax: + 0049 3641 65 6800 E-mail: diersenb@leutra.imb-jena.de
  • M. Kujau
    • Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstraße 11, D-07745 Jena, Germany Fax: + 0049 3641 65 6800 E-mail: diersenb@leutra.imb-jena.de
  • R. Wenderoth
    • Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstraße 11, D-07745 Jena, Germany Fax: + 0049 3641 65 6800 E-mail: diersenb@leutra.imb-jena.de
  • K. Müller
    • Biochemisches Institut der Universität Zürich, Winterthurer Str. 190, CH-8057 Zürich, Switzerland
  • S. Matzku
    • Merck KGaA, Med Chem Bioorganica, Frankfurter Straße 250, D-64271 Darmstadt, Germany
  • A. Plückthun
    • Biochemisches Institut der Universität Zürich, Winterthurer Str. 190, CH-8057 Zürich, Switzerland
  • D. Riesenberg
    • Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstraße 11, D-07745 Jena, Germany Fax: + 0049 3641 65 6800 E-mail: diersenb@leutra.imb-jena.de
ORIGINAL PAPER

DOI: 10.1007/s002530050855

Cite this article as:
Horn, U., Strittmatter, W., Krebber, A. et al. Appl Microbiol Biotechnol (1996) 46: 524. doi:10.1007/s002530050855

Abstract

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl−1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-termi-nal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hok/sok suicide system, improving plasmid maintenance, and the inducible lac p/o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l−1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fed-batch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 g l−1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

Copyright information

© Springer-Verlag Berlin Heidelberg 1996