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Cited by 99Protein purification and stabilityMicrobial Metabolic Engineering and BioproductionBacterial Genetics and Biotechnology

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media

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Gąciarz, Anna, Khatri, Narendar Kumar, Velez-Suberbie, M. Lourdes, Saaranen, Mirva J., Uchida, Yuko, Keshavarz-Moore, Eli, Ruddock, Lloyd W.·2017·Microbial Cell Factories
DOI 10.1186/s12934-017-0721-xPMC PMC5471842
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Abstract

The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

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  • CyDisCo, preserving the native cytoplasm environment, is amenable to large-scale cultivation in chemically defined minimal media, as demonstrated by a high-expression yield of 139 mg/L of an IgA1 scFv.

    “Additionally, unlike the ΔtrxB/Δgor strains (SHuffle or Origami), CyDisCo, preserving the native cytoplasm environment, results also amenable to large-scale cultivation in chemically defined minimal media as demonstrated by the high-expression yield (139 mg/L) of the scFv of an IgA1 [55].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [55]

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Authors
Gąciarz, A., Khatri, N. K., Velez-Suberbie, M. L., Saaranen, M. J., Uchida, Y., Keshavarz-Moore, E., & Ruddock, L. W.
Affiliations
University of Oulu (FI)
Corresponding author
Ruddock, Lloyd W.
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2017-06-15
Volume
16
Issue
1
Pages
108-108
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/s12934-017-0721-x
PMC
PMC5471842
OpenAlex
W2627092082
Cited by
99
References
2
Citations by year
2026: 9 · 2025: 7 · 2024: 13 · 2023: 17 · 2022: 12 · 2021: 15 · 2020: 12 · 2019: 9 · 2018: 5
FWCI
2.91
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • UCL Discovery (University College London) (submittedVersion)
Funders
  • Suomen Akatemia
  • Sigrid Juséliuksen Säätiö
  • Biocenter Oulu
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
soluble protein expression, cytoplasmic expression, Escherichia coli, sulfhydryl oxidase, disulfide bond isomerase, fed-batch fermentation, interleukin-6, glycerol carbon source
MeSH terms
Animals, Avidin, Inclusion Bodies, Chickens, Culture Media, Cytoplasm, Disulfides, Escherichia coli, Female, Fermentation, Glucose, Glycerol, Humans, Immunoglobulin Fragments, Oxidation-Reduction, Recombinant Proteins, Interleukin-6, Bioreactors, Human Growth Hormone, Escherichia coli Proteins

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