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Cited by 25Protein purification and stabilityMonoclonal and Polyclonal Antibodies ResearchBacterial Genetics and Biotechnology

Efficient antibody assembly in E. coli periplasm by disulfide bond. Folding factor Co-expression and culture optimization

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Rodriguez, Carlos, Nam, Dong Hyun, Kruchowy, Evan, Ge, Xin·2017·Applied Biochemistry and Biotechnology
DOI 10.1007/s12010-017-2502-8PMC PMC5898612
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Abstract

Molecular chaperones and protein folding factors of bacterial periplasmic space play important roles in assisting disulfide bond formation and proper protein folding. In this study, effects of disulfide bond protein (Dsb) families were investigated on assembly of 3F3 Fab, an antibody inhibitor targeting matrix metalloproteinase-14 (MMP-14). By optimizing DsbA/C co-expression, promoter for 3F3 Fab, host strains, and culture media and conditions, a high yield of 30-mg purified 3F3 Fab per liter culture was achieved. Produced 3F3 Fab exhibited binding affinity of 34 nM and inhibition potency of 970 nM. This established method of DsbA/C co-expression can be applied to produce other important disulfide bond-dependent recombinant proteins in E. coli periplasm.

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  • A periplasmic recovery of 30 mg/L of functional Fab was reported using pelB and induction with 0.1 mM IPTG at 30 °C overnight when coexpressed with DsbA/C chaperones in shake flasks.

    “A similar periplasmic recovery (30 mg/L) of functional Fab was reported using pelB and induction with 0.1 mM IPTG at 30 °C o.n. when coexpressed with the DsbA/C chaperones in shake flasks [88].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [88]

    The Results section, “30 mg/L 3F3 Fab Yielded in Rich Culture Media” (page unavailable), reports 30 mg of purified Fab per liter at 30 °C. The Methods section, “3F3 Fab Expression” (page unavailable), describes pelB-based expression and overnight induction with 0.1 mM IPTG. However, the 30 mg/L condition used EnBase medium and co-expression with DsbA alone, not both DsbA and DsbC; our copy does not identify it as a shake-flask yield. (LLM, conf=0.89)

    Experimental — accuracy not yet measured

  • Rodriguez reported a significant improvement in functional Fab expression into the E. coli periplasm (30 mg/L) as a result of coexpression with wild type periplasmic DsbA/C.

    “In this regard, Rodriguez and coworkers have reported a significant improvement in functional Fab expression into the E. coli periplasm (30 mg/L) as a result of its coexpression with the wild type periplasmic DsbA/C [88].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [88]

    The Results section, “30 mg/L 3F3 Fab Yielded in Rich Culture Media” (page unavailable), reports 30 mg of purified Fab per liter. But Materials and Methods, “3F3 Fab Expression” (page unavailable), specifies that the optimized EnBase culture carried pBAD-DsbA, not a plasmid co-expressing both DsbA and DsbC. The claim gets the yield right but incorrectly attributes it to co-expression with both factors. (LLM, conf=0.89)

    Experimental — accuracy not yet measured

  • The synergistic effect of DsbA/DsbC has been successfully assessed as an effective way to improve the soluble expression of Fabs and scFvs in the E. coli periplasmic space.

    “The synergistic effect of DsbA/DsbC has been next successfully assessed as an effective way to improve the soluble expression of Fabs [56,88] and scFvs [170] in the E. coli periplasmic space.”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [88]

  • The 22 amino acids long PelB signal sequence from Erwinia carotovora is the most frequently used for transportation of Fabs and scFvs.

    “The 22 amino acids long PelB (pectate lyase B) signal sequence from Erwinia carotovora [157] is the most frequently used for transportation of the Fabs [88,96] and scFvs [30,101,158,159].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [88]

    Citation [157] is attached to the description of PelB as a 22-amino-acid signal sequence from Erwinia carotovora. The Materials and Methods section (page unavailable) says the authors used pelB leader peptides for a Fab expression vector, but our copy does not state the sequence length or organism of origin. Searches for those details found no supporting passage. (LLM, conf=0.73)

    Experimental — accuracy not yet measured

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Cites

No references could be resolved yet.

Research works printed but not linked: 26

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Cited by

Sorted by citations

Authors
Rodriguez, C., Nam, D. H., Kruchowy, E., & Ge, X.
Affiliations
University of California, Riverside (US)
Corresponding author
Ge, Xin
ORCID
Venue
Applied Biochemistry and Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2017-05-10
Volume
183
Issue
2
Pages
520-529
ISSN
  • 0273-2289 (ISSN-L)
  • 1559-0291
DOI
10.1007/s12010-017-2502-8
PMC
PMC5898612
OpenAlex
W2613086300
Cited by
25
References
0
Citations by year
2026: 2 · 2025: 1 · 2024: 3 · 2023: 5 · 2022: 2 · 2021: 4 · 2020: 3 · 2019: 2 · 2018: 1 · 2017: 2
FWCI
0.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Applied Biochemistry and Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • Division of Chemical, Bioengineering, Environmental, and Transport Systems (1453645)
  • National Institute of General Medical Sciences (R01GM115672)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
antibody assembly, Fab fragment, DsbA, DsbC, Escherichia coli, MMP-14, recombinant antibody production, culture condition optimization, promoter optimization, host strain selection, binding affinity
MeSH terms
Escherichia coli, Humans, Immunoglobulin Fab Fragments, Recombinant Proteins, Protein Folding, Protein Disulfide-Isomerases, Periplasm, Escherichia coli Proteins

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