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

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.

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Acknowledgments

We thank Andreas Plückthun for providing the plasmids pAK100, pHB110. Thanks to Gisela Sudermann and Liane Schlenckert for excellent technical support. Special thanks to Giles Johnson and Friedrich Kring for critical reading of the manuscript. This work was funded by Merck KGaA Darmstadt, Germany.

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Correspondence to Uwe Horn.

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Kraft, M., Knüpfer, U., Wenderoth, R. et al. A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of Escherichia coli . Appl Microbiol Biotechnol 76, 1413–1422 (2007). https://doi.org/10.1007/s00253-007-1121-7

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Keywords

  • Human Epidermal Growth Factor Receptor
  • Misfolded Protein
  • Leader Peptide
  • Luciferase Signal
  • Cloning Technology