Cited by 131Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityMicrobial Metabolic Engineering and Bioproduction

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

Open access
Robinson, Michael-Paul, Ke, Na, Lobstein, Julie, Peterson, Cristen, Szkodny, Alana, Mansell, Thomas J., Tuckey, Corinna, Riggs, Paul D. +5 more·2015·Nature Communications

Abstract

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals’—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.

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  • E. coli SHuffle®T7 Express is useful for the expression of complex proteins containing disulfide bonds.

    “The usefulness of E. coli SHuffle®T7 Express for the expression of complex proteins containing disulfide bonds has already been demonstrated [6, 22, 23].”

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  1. Hakim R. & Benhar I. ‘Inclonals': IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system. MAbs 1, 281– 287 (2009). 20065645 PMC2726588

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Authors
Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T. J., Tuckey, C., Riggs, P. D., Colussi, P. A., Noren, C. J., Taron, C. H., DeLisa, M. P., & Berkmen, M.
Affiliations
  • Cornell University (US)
  • New England Biolabs (United States) (US)
Corresponding author
  • DeLisa, Matthew P.
  • Berkmen, Mehmet
Venue
Nature Communications
Published
2015-08-27
Volume
6
Issue
1
Pages
8072-8072
ISSN
2041-1723 (ISSN-L)
OpenAlex
W1239799147
Cited by
131
References
52
Citations by year
2026: 5 · 2025: 13 · 2024: 6 · 2023: 13 · 2022: 11 · 2021: 17 · 2020: 20 · 2019: 10 · 2018: 12 · 2017: 15 · 2016: 7 · 2015: 1
FWCI
9.44
Type
journal article
Language
en
Open access
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • National Science Foundation
  • National Institutes of Health (1r41ai092969-01a1)
  • National Institute of Allergy and Infectious Diseases (AI092969-01A1, R41 AI092969)
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 › Microbial Metabolic Engineering and Bioproduction
Keywords
IgG, Escherichia coli, disulfide bond stability, Fcγ receptor binding, effector function, membrane translocation
MeSH terms
Antibodies, Antibody Formation, Bacteriophages, Cytoplasm, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Glycosylation, Immunoglobulin G, Plasmids, Blotting, Western, Surface Plasmon Resonance, Protein Transport, Organisms, Genetically Modified

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