Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Escherichia coli Cytoplasmic Expression of Disulfide-Bonded Proteins: Side-by-Side Comparison between Two Competing Strategies

  • Angel Castillo-Corujo (Faculty of Biochemistry and Molecular Medicine, University of Oulu) ;
  • Yuko Uchida (Faculty of Biochemistry and Molecular Medicine, University of Oulu) ;
  • Mirva J. Saaranen (Faculty of Biochemistry and Molecular Medicine, University of Oulu) ;
  • Lloyd W. Ruddock (Faculty of Biochemistry and Molecular Medicine, University of Oulu)
  • Received : 2023.11.17
  • Accepted : 2024.03.18
  • Published : 2024.05.28
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Abstract

The production of disulfide bond-containing recombinant proteins in Escherichia coli has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of E. coli: i) engineered strains with deletions in the disulfide reduction pathways, e.g. SHuffle, and ii) the co-expression of oxidative folding catalysts, e.g. CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.

Keywords

Acknowledgement

The use of the facilities of the Biocenter Oulu core facilities, a member of Biocenter Finland, is gratefully acknowledged. We also gratefully acknowledge provision of plasmids by Heli I. Alanen and Jenni Limnell.

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