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Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

Susan Schlegel1, Edurne Rujas1, Anders Jimmy Ytterberg2, Roman A Zubarev2, Joen Luirink3 and Jan-Willem de Gier1*

  • * Corresponding author: Jan-Willem de Gier

  • † Equal contributors

Author Affiliations

1 Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm SE-106 91, Sweden

2 Chemistry I Division, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

3 Section of Molecular Microbiology, Department of Molecular Cell Biology, VU University, De Boelelaan 1085, Amsterdam, 1081 HV, The Netherlands

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Microbial Cell Factories 2013, 12:24  doi:10.1186/1475-2859-12-24

Published: 12 March 2013



In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled.


Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized.


Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment.

Recombinant protein; Protein production; Escherichia coli; Lemo21(DE3); Protein translocation; Periplasm; Sec-translocon