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Cited by 100Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityBiochemical and Structural Characterization

Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli

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Gaciarz, Anna, Veijola, Johanna, Uchida, Yuko, Saaranen, Mirva J., Wang, Chunguang, Hörkkö, Sohvi, Ruddock, Lloyd W.·2016·Microbial Cell Factories
DOI 10.1186/s12934-016-0419-5PMC PMC4727284
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Abstract

BACKGROUND: Disulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations. RESULTS: Here we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active. CONCLUSIONS: Our results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.

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Authors
Gaciarz, A., Veijola, J., Uchida, Y., Saaranen, M. J., Wang, C., Hörkkö, S., & Ruddock, L. W.
Affiliations
  • University of Oulu (FI)
  • Oulu University Hospital (FI)
Corresponding author
Ruddock, Lloyd W.
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2016-01-25
Volume
15
Issue
1
Pages
22-22
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/s12934-016-0419-5
PMC
PMC4727284
OpenAlex
W2266449668
Cited by
100
References
1
Citations by year
2026: 8 · 2025: 7 · 2024: 9 · 2023: 12 · 2022: 14 · 2021: 13 · 2020: 10 · 2019: 11 · 2018: 8 · 2017: 6 · 2016: 1
FWCI
7.52
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Biotieteiden ja Ympäristön Tutkimuksen Toimikunta
  • Sigrid Juséliuksen Säätiö
  • Biocenter Oulu
  • Sydäntutkimussäätiö
Topics
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
antibody fragments, scFv, Fab fragment, disulfide bond formation, disulfide bond isomerization, cytoplasmic expression, Escherichia coli, protein folding
MeSH terms
Animals, Antibodies, Cytoplasm, Escherichia coli, Genetic Vectors, Humans, Immunoglobulin Fragments, Molecular Weight, Plasmids, Solubility, Protein Engineering, Spectrometry, Mass, Electrospray Ionization, Mice, Single-Chain Antibodies

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