Applied Genetics and Molecular Biotechnology

Applied Microbiology and Biotechnology

, Volume 76, Issue 6, pp 1413-1422

First online:

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

  • Mario KraftAffiliated withDepartment Bio Pilot Plant, Leibniz-Institute for Natural Product Research and Infection Biology-Hans-Knöll-Institute
  • , Uwe KnüpferAffiliated withDepartment Bio Pilot Plant, Leibniz-Institute for Natural Product Research and Infection Biology-Hans-Knöll-Institute
  • , Rolf WenderothAffiliated withDepartment of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product Research and Infection Biology-Hans-Knöll-Institute
  • , André KacholdtAffiliated withInstitute for Farm Animals Sciences and Technology (NTT), University of Rostock
  • , Patricia PietschmannAffiliated withDepartment Expression Systems and Cell Supply, Merck KGaA
  • , Björn HockAffiliated withDepartment Expression Systems and Cell Supply, Merck KGaA
  • , Uwe HornAffiliated withDepartment Bio Pilot Plant, Leibniz-Institute for Natural Product Research and Infection Biology-Hans-Knöll-Institute Email author 

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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.