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Cited by 41Protein purification and stabilityMicrobial Metabolic Engineering and BioproductionMonoclonal and Polyclonal Antibodies Research

Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

Open access
Fink, Mathias, Vazulka, Sophie, Egger, Esther, Jarmer, Johanna, Grabherr, Reingard, Cserjan‐Puschmann, Monika, Striedner, Gerald·2019·Biotechnology Journal
DOI 10.1002/biot.201800637
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Abstract

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome‐integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon‐limited microbioreactor cultivations with yields of 0–7.4 mg Fab per gram of cell dry mass. Antigen‐binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid‐free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80–300% of plasmid‐based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.

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  • The OmpA-leader sequence was selected as the best in plasmid-free expression systems for the production of a functional Fab fragment.

    “Recently, the OmpA-leader sequence (MKKTAIAIAVALAGFATVAQA) was also selected as the best in plasmid-free expression systems (GI) for the production of a functional Fab fragment [74,104].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [104]

    Section 3.1 and Table 1 describe genome-integrated, plasmid-free systems using OmpA to produce Fab fragments. But Section 3.3.1 says Fab yields with DsbA were not clearly different from those with OmpA and that the authors could not generally conclude which translocation route was favorable; it does not support selecting OmpA as the best leader sequence. Page numbers were not available in our copy. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

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Authors
Fink, M., Vazulka, S., Egger, E., Jarmer, J., Grabherr, R., Cserjan‐Puschmann, M., & Striedner, G.
Affiliations
  • BOKU University (AT)
  • Boehringer Ingelheim (Austria) (AT)
Corresponding author
Cserjan‐Puschmann, Monika
ORCID
Venue
Biotechnology Journal
Publisher
Wiley
Published
2019-07-26
Volume
14
Issue
11
Pages
e1800637-e1800637
ISSN
  • 1860-6768 (ISSN-L)
  • 1860-7314
DOI
10.1002/biot.201800637
OpenAlex
W2953957022
Cited by
41
References
1
Citations by year
2026: 6 · 2025: 3 · 2024: 7 · 2023: 4 · 2022: 8 · 2021: 6 · 2020: 5 · 2019: 2
FWCI
1.74
Type
journal article
Language
en
Open access
Licence
Locations
  • Biotechnology Journal (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Bundesministerium für Digitalisierung und Wirtschaftsstandort
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
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
Keywords
Fab fragment, Escherichia coli, antigen-binding region, translocation mechanism, gene copy number, Escherichia coli BL21(DE3), recombinant protein yield
MeSH terms
Biotechnology, Escherichia coli, Genetic Vectors, Immunoglobulin Fab Fragments, Kinetics, Plasmids, Prospective Studies, Recombinant Proteins, Gene Expression Regulation, Bacterial, Genome, Bacterial, Gene Dosage, Bioreactors, Protein Sorting Signals

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