Applied Microbiology and Biotechnology

, Volume 76, Issue 6, pp 1413–1422

A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of Escherichia coli

  • Mario Kraft
  • Uwe Knüpfer
  • Rolf Wenderoth
  • André Kacholdt
  • Patricia Pietschmann
  • Björn Hock
  • Uwe Horn
Applied Genetics and Molecular Biotechnology
  • 272 Downloads

Abstract

The functional analysis of individual proteins or of multiprotein complexes—since the completion of several genome sequencing projects—is in focus of current scientific work. Many heterologous proteins contain disulfide-bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degP-promoter-based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies.

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Mario Kraft
    • 1
  • Uwe Knüpfer
    • 1
  • Rolf Wenderoth
    • 2
  • André Kacholdt
    • 3
  • Patricia Pietschmann
    • 4
  • Björn Hock
    • 4
  • Uwe Horn
    • 1
  1. 1.Department Bio Pilot PlantLeibniz-Institute for Natural Product Research and Infection Biology-Hans-Knöll-InstituteJenaGermany
  2. 2.Department of Molecular and Applied MicrobiologyLeibniz-Institute for Natural Product Research and Infection Biology-Hans-Knöll-InstituteJenaGermany
  3. 3.Institute for Farm Animals Sciences and Technology (NTT)University of RostockRostockGermany
  4. 4.Department Expression Systems and Cell SupplyMerck KGaADarmstadtGermany

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