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

Microbial Cell Factories

Research

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

[1] Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91, Stockholm, Sweden;Chemistry I Division, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden;Section of Molecular Microbiology, Department of Molecular Cell Biology, VU University, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands;

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

DOI  :  10.1186/1475-2859-12-24

 received in 2013-01-10, accepted in 2013-03-05,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundIn 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.ResultsBoth 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.ConclusionsSaturating 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.

【 授权许可】

Unknown   
© Schlegel et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.